A glovebox-integrated confocal microscope for quantum sensing in inert atmosphere

Quantum technology is a rapidly developing field with a broad range of applications in science and technology. It applies the fundamental principles of quantum mechanics, where effects become significant and observable only at the nano-scale level. Recent developments in quantum technology have greatly advanced spectroscopy, providing exceptional precision in measuring energy levels, magnetic fields, and molecular dynamics, as well as ultra-sensitive detection capabilities. Spectroscopy techniques allow detailed study of material properties and their behavior, providing critical insights into chemical composition and electronic structure. In particular, confocal microscopy combined with nitrogen-vacancy (NV) centers in diamond has become a powerful tool for studying materials. NV centers offer high sensitivity and spatial resolution for detecting extremely weak magnetic and electric fields, making it a valuable quantum sensor.

The NV center is a point defect in the diamond crystal lattice that consists of a nitrogen atom substituting one of the carbon atoms and an adjacent vacancy. It can exist in a neutral charge state (NV⁰), or it can attract an additional electron to achieve a more stable electron configuration forming a negatively charged NV⁻ state.¹ For simplicity, negatively charged nitrogen-vacancy centers will be referred to as NV centers.

Several key properties of NV centers are important for nano-scale sensing. First, it has spin-dependent fluorescence. The NV center energy structure is characterized by triplet ground and excited states with an electron spin S = 1 and an intermediate singlet state with S = 0. After laser-induced excitation, the NV center has a preferential direct relaxation into the m_s = 0 spin state with bright fluorescence (>600 nm), while the m_s = ±1 states are more likely to pass through a metastable singlet state and decay to the ground state non-radiatively. This spin dependency allows optical spin polarization of the NV center into the m_s = 0 state. Second, the NV center’s spin state can be manipulated by microwave (MW) pulses, setting it into either m_s = ±1 states or in a superposition of states. A more detailed description of the NV center properties can be found in Ref. 1. Finally, the sensing volume of a single NV center depends on the sensing protocol, the implantation depth, and the NV center orientation.^2,3 For successful detection, the investigated material (for example, nuclear spins in a molecule) must be in close proximity of the NV center. One of the approaches to achieve this is to use a shallowly implanted NV center and deposit or functionalize the investigated material on the diamond surface. By placing the material close to the NV center, the sensor can detect subtle changes in magnetic fields or other quantum properties at a very small scale, providing insights into the material's structure or behavior.

Nuclear spins in the presence of external magnetic field oscillate at the Larmor frequency, creating an alternating magnetic noise. This nanoscale magnetic noise can be detected by an NV center,^4,5 allowing performing nuclear magnetic resonance (NMR) spectroscopy at the level of a single molecule⁶ or for sensing single nuclear spins.⁷ The NV center-based NMR techniques do not require large sample volumes or high magnetic fields and can operate under ambient conditions.⁸ This feature opens great possibilities for studying materials and biological systems with high resolution and sensitivity. Moreover, successful detection of nuclear spin arrays on the diamond surface can be a first step toward the realization of an NV center-based quantum simulator, where external spins are used as qubits and NV centers serve for initialization, manipulation, and read-out.^9,10

Among the materials studied or proposed for NV center-based NMR spectroscopy, many exhibit either air or humidity sensitivity and can alter their properties upon air exposure. For instance, transition metal dichalcogenides (TMDs) are promising candidates to create ultrathin uniform films.¹¹ However, they can degrade upon air exposure.^12,13 Proteins are also commonly studied using NV centers, but some of them are prone to oxidation.¹⁴ These and other sensitive materials might degrade, aggregate, or decompose over time due to exposure to oxygen or moisture. Such reactions can be accelerated by light exposure¹⁵ or by increased temperatures caused, for example, by laser illumination.¹⁶ 

One interesting air-sensitive material is black phosphorus (BP).¹⁷ Phosphorus isotope ³¹P is a good candidate for the NV center-based spin detection thanks to its 100% natural abundance and a high gyromagnetic ratio γ = 1.725 kHz/G. Shallow NV centers tend to have a short coherence time (T₂) due to interaction with spin impurities on the surface. Some of the quantum sensing protocols, for example, dynamical decoupling sequence, have limited sensing time, defined by NV center’s spin decoherence period.¹⁸ High gyromagnetic ratio of the phosphorus and nuclear spin-$12$ allows detection of BP spins at low magnetic fields, making NV centers even with short coherence time effective sensing probes.

There are several techniques to protect air-sensitive materials such as BP by surrounding or coating them with a stable material, also known as encapsulation. For example, hexagonal boron nitride (hBN) is widely used for shielding two-dimensional (2D) materials from contamination.¹⁹ In this approach, a layer of hBN is transferred on top of the 2D material, sealing it between the substrate and the hBN flake itself [Fig. 1(a), left]. However, this technique has several disadvantages in NV center-based applications. First, it introduces non-target spins into the system with shallow NV centers:²⁰ boron isotopes ¹⁰B and ¹¹B and nitrogen isotopes ¹⁴N and ¹⁵N. Magnetic noise from these spins can affect shallow NV centers and may result in a spurious signal in the measured data.²¹ Second, negatively charged boron vacancies present in hBN have fluorescence that overlaps with NV center fluorescence, making both defects optically indistinguishable [Fig. 1(b)].²² 

Another commonly used approach for protection from ambient conditions is glass encapsulation. In this method, a glass cover slip is placed over the diamond sample with a BP flake and then sealed with epoxy using ultraviolet light [Fig. 1(a), right]. This encapsulation unavoidably creates an air–glass interface between the objective of the confocal microscope and the diamond with NV centers, thus reducing both laser excitation intensity and fluorescence collection efficiency [Fig. 1(c)].

Other encapsulation techniques²³ have similar disadvantages: either non-target spins from the shielding material will be present in the sensing area of the NV center, or there will be multiple layers of different environments with varying refractive indices causing light scattering, reflection, and absorption. In many cases, to avoid air exposure of the sample, measurements are preferably performed inside a glovebox with an inert environment.

Since available shielding techniques do not allow NV center-based NMR spectroscopy, our solution is to integrate the glovebox in an existing home-built confocal microscope. A glovebox-integrated confocal microscope would allow not only NV center-based spin sensing experiment with the BP but also enable a variety of experiments in a controlled environment free from humidity, oxygen, and other contaminants.

The modified custom-made confocal microscope follows a conventional configuration, commonly used for experiments with NV centers.^24,25 A schematic of the experimental setup is shown in Fig. 2(a). The diode laser produces continuous and pulsed light at a wavelength of 518 nm, which is subsequently coupled to fiber and emitted through a collimator. The laser beam is attenuated with a neutral density filter (NDF) and then passes through a short-pass filter (F₁) to remove light from any potential dispersion of the optical fiber. A beam sampler (BS) guides the green light toward the objective, which focuses it on the sample. A rough focus is achieved manually on an XYZ mechanical stage and then precise focus is achieved via a piezo controlled stage. The emitted fluorescence is collected by the same objective, passes through the beam sampler, and is focused on a pinhole (PH). The pinhole acts as a spatial filter by removing any emission that is not originating from the illuminated focal spot. Unlike the widefield microscope, this configuration of the confocal microscope allows us to perform depth scans of the sample and achieves higher spatial resolution. Depending on the measurement, after the pinhole, the fluorescence is either guided toward the optical detector (APD) or toward the spectrometer. In both cases, a long-pass filter (F₂ or F₃ respectively) is used to remove the laser emission. The APD signal goes either to the data acquisition system or to the photon fast counter. All measurements are controlled and analyzed with Qudi software.²⁶ 

To perform NV center spin manipulation, we use either a continuous wave MW source or an arbitrary waveform generator (AWG) for MW pulses [Fig. 2(b)]. The MW signal is amplified and delivered in the NV centers’ proximity by a 20 μm diameter copper (99.99% purity) wire that is placed on the diamond surface and soldered to the sample holder. A permanent magnet is installed on a holder with four degrees of freedom above the sample stage [Fig. 3(a)]. Precise magnetic field alignment allows us to reach magnetic field projection on the NV center axis of up to 1000 G. All the equipment used for the presented confocal microscope is described in Table I.

The glovebox is made of polymethyl methacrylate (PMMA, acryl glass) with dimensions of 900 × 500 × 500 mm³ with 10 mm thickness and is manufactured by Sylatech GmbH, Germany. It provides enough space to install the sample holder, perform a manual focusing, and align the magnetic field [Fig. 3]. The small internal volume allows us to reach an inert atmosphere within several hours. In the pumped glovebox, the environment is very dry (3%–4% relative humidity) because, in our case, the nitrogen gas removes moisture and oxygen. The inert atmosphere maintains moderate temperature fluctuations and prevents

Unwanted side reactions include oxidation, hydrolysis, and other degradation processes. For oxygen control, an oxygen analyzer (Oxy.IQ from General Electric Company, USA) is used, and temperature and humidity levels are monitored with a digital thermo-hygrometer from Dostmann.

There are two main challenges associated with the glovebox-integrated confocal microscope. These challenges involve leaks that can contaminate inert atmosphere and pressure instability that will induce vibrations of optical elements.

The main source of potential leaks is the hole made to pass the objective inside the glovebox. To counter the potential leaks, we isolated the objective with synthetic rubber, which was glued to the glovebox. Aluminum tape serves as an additional protection from leaks on the glovebox side. Rubber rings and parafilms create better isolation at the end of the objective [Fig. 3(d)]. A continuous flow of inert gas maintains the atmosphere and compensates for leaks from the sealed inlets.

To guarantee the vibration-free setup, we ensured to keep all optical elements of the excitation and detection parts outside the glovebox, allowing versatile measurements and ensuring stability. The automatic outlet valve opens at overpressure of 2–5 mbar. Maintaining positive pressure is necessary to protect air sensitive samples from ambient air. However, because the objective is mounted on the piezo stage and is isolated with synthetic rubber, it is sensitive to any pressure changes within the glovebox. To stabilize the pressure, an additional bleeding valve with ball protection was added [Fig. 3(c)]. Ball protection is necessary when negative pressure may occur during sample manipulation or magnetic field alignment in the glovebox.

Overall, we observed that our setup is successfully capable of maintaining an inert environment. In the equilibrated state, the glovebox can reach oxygen level as low as 14 ppm. With a higher flow of nitrogen, it is possible to further reduce the O₂ concentration to below 10 ppm; however, this would require modifications in the pressure-stabilizing outlet. Observed humidity levels reach 3%–4% and stabilize at those levels.

To demonstrate the capability of the glovebox-integrated confocal microscope, we performed two experiments. The first experiment with an air-sensitive BP flake demonstrates that the glovebox achieves sufficiently low oxygen levels to conduct experiments with air-sensitive materials. The second experiment demonstrates that it is possible to detect NV centers and manipulate their electron spins under the transferred BP flake.

All fluorescence images presented in this paper were taken at 100 μW laser excitation power using an air objective with NA = 0.95 (Table I).

A thin flake was exfoliated from a BP crystal (HQ Graphene, Netherlands) inside an industrial glovebox from Vigor, USA (argon gas, oxygen 0.1 ppm, moisture 1.1 ppm) and transferred on a silicon substrate using a dry transfer method.²⁷ The sample was then delivered to the glovebox-integrated confocal microscope in a sealed transport box. We ensured low oxygen exposure, keeping the box sealed until the oxygen level dropped below 40 ppm.

Figure 4(a) shows the fluorescence image of the BP flake inside the glovebox-integrated confocal microscope in a nitrogen environment. A typical spectrum of the bright area [Fig. 4(a)] has a peak at around 1070 nm or 1.16 eV (see the supplementary material), which is consistent with an emission of a bilayer BP.²⁸ These data align well with the fact that only mono- and bi-layers of BP have emission in the near infrared spectrum detectable by the APD. Crucially, we repeated the spectral measurements across 30 min and found no changes in the fluorescence image. This confirms the robust optical quality of the flake and that no degradation has occurred.

It has been reported that BP is susceptible to laser-induced oxidation, a property that has even been used to achieve phosphorene layers.²⁹ To further validate the stability of the BP flake in the glovebox-integrated confocal microscope, we exposed the sample to the ambient conditions and recorded the spectrum in real time. Similar to Ref. 29, under simultaneous laser and air exposure, induced oxidation occurs on the bilayer and bulk areas of the BP. As a result, the bilayer thins down to a monolayer, as confirmed by its fluorescence spectrum, which showed a peak at around 740 nm (1.67 eV), and further damage eventually creates a hole in the flake [Fig. 4(c)], which was also observed using atomic force microscopy (see the supplementary material). Irreversible damage occurs within several minutes, even in the thicker areas of the BP flake [Fig. 4(b)].

A scanning clock frequency of 300 Hz allows us to take fluorescence images during air exposure without inducing visible degradation of the BP flake. However, prolonged laser exposure on a single spot of the flake results in significant damage, indicating that long-term measurements with NV center-based spin sensing would not be possible under ambient conditions. The same measurements performed in our glovebox with an inert atmosphere did not show any degradation of the BP flake, highlighting the importance of an inert environment for successful study of air-sensitive materials under prolonged laser exposure.

For measurements with NV centers, we used a type IIa diamond plate with dimensions 2 × 2 × 0.5 mm³ and (100) surface orientation produced by Element Six Ltd., United Kingdom. Shallow NV centers were implanted by Diatope GmbH, Germany. Implantation of ¹⁵N⁺ with dose $10915N+cm2$ was performed at an energy of 2 keV, followed by annealing the diamond sample in ultra-high vacuum at 1000 °C for 2 h for vacancies to diffuse and to form NV centers at approximate depth of 3.7 nm.³⁰ 

The transfer and transportation of the BP flake on the diamond substrate were done analogically to the sample on the silicon substrate described in Sec. III A. After confirming that the transferred BP flake was thin enough to allow NV centers to be visible through it [Fig. 5(a)] and did not have a detectable by the APD emission [Fig. 5(b)], the sample was exposed to air for 20 minutes for wire installation and consequent overnight glovebox pumping. We observed by comparison of fluorescence images that during this time, the BP flake did not visibly degrade, even in the area with a monolayer phosphorene.

To demonstrate that the proximity to the BP flake does not affect the properties of NV centers, we performed the typical experiments for quantum sensing with NV centers such as optically detected magnetic resonance (ODMR), measuring Rabi oscillations, and determined the electron spin coherence time T₂ [Figs. 5(c)–5(e)]. It was possible to focus on a single NV center under the BP flake of approximately 3–5 layers in thickness. The NV center’s fluorescence did now show any jumps, thus suggesting a stable charge state comparable with other shallow implanted samples.

Splitting of the ODMR line indicates a small applied magnetic field of 30 G. The observed linewidth of 20 MHz and an average contrast of 13% in the resonance dips demonstrate favorable conditions for NV center spin manipulation. The measurable Rabi oscillation frequency of 25 MHz further confirms the effective control over the NV centers' spin dynamics. Hahn echo measurements showed either short coherence time with an average value of 1.57 µs or longer T₂ with signal modulations caused by the coupling of the electron spin and the ¹⁵N nuclear spin of the NV center due to misalignment of the magnetic field [Fig. 5(e)].^21,31

From the observed data, we can conclude that the presence of the BP flake on the diamond surface does not have a negative impact on shallow NV centers. This suggests that phosphorus spin detection using NV centers is feasible, allowing the measurement of its properties for potential qubit applications. The high gyromagnetic ratio of phosphorus nucleus would allow detecting it using NV centers at a magnetic field above 250 G, given the observed coherence times.³² 

The glovebox-integrated confocal microscope presented here is a useful tool for NV center-based NMR spectroscopy of air-sensitive materials. Thanks to its simplicity, the glovebox system does not require any expensive elements and its maintenance is straightforward. NV centers are frequently used to investigate various materials,¹² polymers and biological molecules,^3,20 many of which alter their properties when exposed to air or moisture. With this setup, it is possible to study these materials under controlled oxygen levels in the glovebox atmosphere.

Despite the demonstrated advantages of the setup, there are several limitations that should be taken into consideration.

Wire installation (Secs II and III A) is a necessary step to deliver MW field to NV centers, but during this process, the sample is unavoidably exposed to ambient environment. Modifying the sample holder by adding an MW antenna directly on it³³ or manufacturing one on the diamond surface will be a possible improvement.

As demonstrated with the BP flake, laser exposure in the presence of oxygen induces oxidation, suggesting that the process is analogous to the photobleaching of contaminations on the diamond surface. Oxygen molecules react with surface impurities and adsorbates, decomposing them, which leads to a reduced background fluorescence. However, in an inert environment, this process cannot occur since oxygen levels are insufficient to drive the photochemical oxidation process effectively. This can lead to an accumulation of fluorescing contaminants and adsorbates on the sample surface. In addition, dust and other particles can accumulate on the diamond surface due to the laser focal spot, which can act as optical tweezers, thus causing increased background fluorescence. Therefore, it is important to keep the glovebox environment and sample surfaces clean and to avoid high laser power irradiation.

In some cases, we observed charge instability of NV centers in the nitrogen atmosphere without an increase in background fluorescence. This effect was observed for NV centers both under the BP flake and away from it. These phenomena might be caused by the oxygen desorption³⁴ and requires a further investigation.

Beyond new material research, the controlled atmosphere of the glovebox provides new opportunities to study shallow NV centers and how they are affected by the termination of the diamond surface. There are many studies of various surface terminations and functionalization and their effect on NV centers’ stability,^3,35 where some of them degrade upon exposure to ambient conditions.³⁶ Within the glovebox enclosure, it is possible to create or imitate different kinds of environments and investigate surface band bending and electronic noise to elucidate some of the effects of diamond surface chemistry. A better understanding of shallow NV centers behavior and discovery of optimal ways to improve their properties will enhance their performance in quantum technology applications.

The supplementary material provides a more detailed investigation of the black phosphorus flake and its degradation under air exposure, including fluorescence images, spectra, and an atomic force microscope image with a detailed description of experimental conditions.

We thank Sergei Trofimov from the Helmholtz-Zentrum Berlin for the help with the atomic force microscope images presented in the supplementary material. We acknowledge the German Research Foundation DFG (Project Nos. 410866378 and 410866565) and the German Federal Ministry of Education and Research (BMBF) (DIQTOK No. 16KISQ034) for the financial support.

The authors have no conflicts to disclose.

Kseniia Volkova: Data curation (equal); Formal analysis (equal); Investigation (equal); Project administration (equal); Resources (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). Abhijeet M. Kumar: Investigation (equal); Resources (equal); Validation (equal); Writing – review & editing (equal). Kirill Bolotin: Supervision (equal); Validation (equal); Writing – review & editing (equal). Boris Naydenov: Conceptualization (equal); Funding acquisition (equal); Project administration (equal); Supervision (equal); Validation (equal); Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding authors upon reasonable request.