Chapter 5 - Limits to Resolution of CW STED Microscopy

As one of the most important realizations of stimulated emission depletion (STED) microscopy, the continuous-wave (CW) STED system, constructed by using CW lasers as the excitation and STED beams, has been investigated and developed for nearly a decade. However, a theoretical model of the suppression factors in CW STED has not been well established. In this investigation, the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied. The full-width-at-half-maximum (FWHM) of a CW STED with a doughnut-shaped STED beam is also reanalyzed. It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities. In addition, the FWHM is highly sensitive to both the fluorescence rate (inverse of fluoresce lifetime) and the quenching rate, but insensitive to the rate of vibrational relaxation. For comparison, the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity. Our model is highly consistent with published experimental data for evaluating the spatial resolution. This investigation is important in guiding the development of new CW STED systems.

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.

Nitrogen vacancy (NV) color centers in diamond have useful applications in quantum sensing and fluorescent marking. They can be generated experimentally by ion implantation, femtosecond lasers, and chemical vapor deposition. However, there is a lack of studies of the yield of NV color centers at the atomic scale. In the molecular dynamics simulations described in this paper, NV color centers are prepared by ion implantation in diamond with pre-doped nitrogen and subsequent annealing. The differences between the yields of NV color centers produced by implantation of carbon (C) and nitrogen (N) ions, respectively, are investigated. It is found that C-ion implantation gives a greater yield of NV color centers and superior location accuracy. The effects of different pre-doping concentrations (400–1500 ppm) and implantation energies (1.0–3.0 keV) on the NV color center yield are analyzed, and it is shown that a pre-doping concentration of 1000 ppm with 2 keV C-ion implantation can produce a 13% yield of NV color centers after 1600 K annealing for 7.4 ns. Finally, a brief comparison of the NV color center identification methods is presented, and it is found that the error rate of an analysis utilizing the identify diamond structure + coordination analysis method is reduced by about 7% compared with conventional identification methods.

Nitrogen-vacancy (NV) color center is one kind of luminescent point defect in diamond. NV color center is a composite structure composed of substituted nitrogen atoms and adjacent carbon vacancies in diamond. It can be applied in many fields, such as super-resolution fluorescence imaging, high-sensitive detection, and quantum computing. In order to meet the requirements of NV color center’s applications, many efforts have been devoted to study the manufacturing methods of NV color center. Nowadays, femtosecond (fs) laser technology has been widely used in the field of micro/nanomachining and gradually applied to the manufacturing of diamond NV color centers. In this chapter, the mechanism and characteristics of fs laser micro/nanomachining, the basic properties, and the applications of diamond NV color centers were concisely summarized. Moreover, the ultrafast laser processing of NV color center, the fluorescence detection of NV color center, and the anti-bunching analysis method of single NV color center are introduced and discussed in detail.

Enhanced fluorescence quenching rate of Coumarin 102 in stimulated emission depletion

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

The significant role of telomeres in cells has attracted much attention since they were discovered. Fluorescence imaging is an effective method to study subcellular structures like telomeres. However, the diffraction limit of traditional optical microscope hampers further investigation on them. Recent progress on superresolution fluorescence microscopy has broken this limit. In this work, we used stimulated emission depletion (STED) microscope to observe fluorescence-labeled telomeres in interphase cell nuclei. The results showed that the size of fluorescent puncta representing telomeres under the STED microscope was much smaller than that under the confocal microscope. Two adjacent telomeres were clearly separated via STED imaging, which could hardly be discriminated by confocal microscopy due to the diffraction limit. We conclude that STED microscope is a more powerful tool that enable us to obtain detailed information about telomeres.

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.

In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.

Individual nitrogen vacancy (NV) color centers in diamond are bright, photo-stable, atomic-sized dipole emitters [1]. Consequently, they represent optimal candidates for novel scanning near field microscopy techniques [2]. Here, NV centers form one member of a Forster Resonance Energy Transfer (FRET) pair. Due to their broadband emission (> 100 nm), NVs are versatile donors for FRET to systems absorbing in the near infrared spectral range. Highly-promising applications include, e.g., nanoscale imaging of fluorescent molecules or nanomaterials like graphene [2].

Nitrogen-Vacancy color center in diamond — emerging nanoscale applications in bioimaging and biosensing

Diamond nitrogen vacancy (NV) color centers have good stability at room temperature and long electron spin coherence time, and can be manipulated by lasers and microwaves, thereby becoming the most promising structure in the field of quantum detection. Within a certain range, the higher the concentration of NV color centers, the higher the sensitivity of detecting physical quantities is. Therefore, it is necessary to dope sufficient nitrogen atoms into diamond single crystals to form high-concentration NV color centers. In this study, diamond single crystals with different nitrogen content are prepared by microwave plasma chemical vapor deposition (MPCVD) to construct high-concentration NV color centers. By doping different amounts of nitrogen atoms into the precursor gas, many problems encountered during long-time growth of diamond single crystals under high nitrogen conditions are solved. Diamond single crystals with nitrogen content of about 0.205, 5, 8, 11, 15, 36, and 54 ppm (1 ppm = 10<sup>–6</sup>) are prepared. As the nitrogen content increases, the width of the step flow on the surface of the diamond single crystal gradually widens, eventually the step flow gradually disappears and the surface becomes smooth. Under the experimental conditions in this study, it is preliminarily determined that the average ratio of the nitrogen content in the precursor gas to the nitrogen atom content introduced into the diamond single crystal lattice is about 11. Fourier transform infrared spectroscopy shows that as the nitrogen content inside the CVD diamond single crystal increases, the density of vacancy defects also increases. Therefore, the color of CVD high nitrogen diamond single crystals ranges from light brown to brownish black. Compared with HPHT diamond single crystal, the CVD high nitrogen diamond single crystal has a weak intensity of absorption peak at 1130 cm<sup>–1</sup> and no absorption peak at 1280 cm<sup>–1</sup>. Three obvious nitrogen-related absorption peaks at 1371, 1353, and 1332 cm<sup>–1</sup> of the CVD diamond single crystal are displayed. Nitrogen atoms mainly exist in the form of aggregated nitrogen and single substitutional N<sup>+</sup> in diamond single crystals, rather than in the form of C-defect. The PL spectrum results show that defects such as vacancies inside the diamond single crystal with nitrogen content of 54 ppm are significantly increased after electron irradiation, leading to a remarkable increase in the concentration of NV color centers. The magnetic detection performance of the NV color center material after irradiation is verified, and the fluorescence intensity is uniformly distributed in the sample surface. The diamond single crystal with nitrogen content of 54 ppm has good microwave spin manipulation, and its longitudinal relaxation time is about 3.37 ms.

Nitrogen-vacancy (NV) color centers in diamond have emerged as a focal point in quantum technology research due to their exceptional optical and electronic spin properties. This discovery not only expands our understanding of the material properties of diamond but also paves the way for practical applications in quantum technology. As a solid-state spin quantum system characterized by long electron spin coherence times and stable performance, NV color centers offer distinct advantages in quantum information processing. Recent advancements have enabled the fabrication of high-quality NV color centers with controllable concentration and spatial positioning through techniques such as in situ doping, ion implantation, and hybrid approaches. These technological breakthroughs have significantly enhanced the efficiency of NV center fabrication, thereby establishing a robust foundation for their widespread application in quantum sensing and quantum information technologies. This review provides a compre­hensive introduction to the structure and fundamental properties of NV color centers in diamond, an in-depth analysis of recent progress in their fabrication techniques, and a critical discussion of their current applications in quantum science and technology. Additionally, the challenges, open questions, and future research directions in NV color center studies are addressed.

Enormous potential lies in the weird quantum world for a new generation of information systems that address important and unsolved problems in secure communications, highperformance computing, data storage, and simulation. Surprisingly, single crystal diamond (SCD), long known for its allure as a gemstone, has just the right properties for fabricating critical components that will serve as building blocks of this new quantum technology. A key requirement for useful quantum information devices is the ability to create shared quantum entanglement among large numbers of quantum bits (qubits) independent of their physical locations. This is best done using optical photons, especially for long-distance secure quantum communication applications. However, because photons do not make good quantum memories, a system with long-lived qubits, such as spins and a highfidelity quantum interface to photons would be highly desirable. Although trapped ions are potentially capable of fulfilling this need, there is no substitute for solid-state implementation for ease of scaling and integration. Our approach is to use nitrogen-vacancy (NV) color centers in diamond. These consist of a nitrogen atom paired with a vacancy in the diamond lattice. They provide strong optical transitions that scatter photons efficiently enough to allow individual NVs to be easily visualized with a confocal microscope.1 Unlike most optical emitters, the NV has a ground state with three electron spin sublevels. Proximal nuclear spins provide additional degrees of freedom,2 resulting in numerous choices for qubit encoding. Spin coherence times for such qubits are unusually long, even at room temperature, rivaled only by ion qubits trapped in high vacuum. It is estimated that up to a million single qubit operations can be performed within the NV spin coherence lifetime. Through a fortunate interaction with the singlet excited states, the NV can be spin-polarized to a high degree by illumination with broadband light, such as an LED flashlight, even at room temperature. Equally fortuitous is the fact that one of the electron spin sublevels undergoes an optical cycling transition that is pure enough, at liquid helium temperatures, to allow single-shot spin readout.1 Even at room temperature, this cycling transition is adequate for such a readout if half of the emitted photons can be detected. Encouragingly, our recent theoretical estimates3 and pilot experiments4 with optical plasmon nanowires suggest this might soon be possible. At room temperature, we have demonstrated each of the key elements needed to build few-qubit quantum processors with enough functionality for quantum repeater nodes.5 Specifically, we showed that arbitrary electron spin coherences can be transferred to a nuclear spin where they can be stored for up to a fraction of second and recovered with high fidelity. Furthermore, the stored nuclear spin is sufficiently robust for optical re-initialization cycles of the electron spin, as required for a quantum repeater memory. We also observed multi-qubit interactions between the NV electron spin and nearby nuclear6 and electron7spins up to nanometers away. We are currently in the process of creating long-distance entanglement between pairs of NVs using optical measurements. So far, we have demonstrated narrowband, electrically tunable,8 optical transitions stable enough to allow spontaneous photons, emitted minutes apart, to produce optical interference fringes.9 We have also demonstrated optical Raman spin-flip transitions on one branch of the excited state while simultaneously maintaining an optical cycling transition on the other.10 In summary, we can report surprising progress in realizing scalable quantum information systems with NV color centers in diamond (see Table 1). In the future, we plan to develop large-scale photonic circuits that optically couple numerous NV qubits to construct advanced quantum processors, and highperformance spintronic devices.11

In this paper, we report the enhancement of resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy by a novel method of structured illumination of an excitation beam. Illumination by multiple excitation beams through the specific pupil apertures with high in-plane wave vectors leads to interference of diffracted light flux near the focal plane, resulting in the contraction of the point spread function (PSF) of the excitation. Light spot reduction by the suggested standing wave (SW) illumination method contributes to make up much lower depletion efficiency of the CW STED microscopy than that of the pulsed STED method. First, theoretical analysis showed that the full width at half maximum (FWHM) of the effective PSF on the detection plane is expected to be smaller than 25% of that of conventional CW STED. Second, through the simulation, it was elucidated that both the donut-shaped PSF of the depletion beam and the confocal optics suppress undesired contribution of sidelobes of the PSF by the SW illumination to the effective PSF of the STED system. Finally, through the imaging experiment on 40-nm fluorescent beads with the developed SW-CW STED microscopy system, we obtained the result which follows the overall tendency from the simulation in the aspects of resolution improvement and reduction of sidelobes. Based on the obtained result, we expect that the proposed method can become one of the strategies to enhance the resolution of the CW STED microscopy.