Fig. 2 a,b,c show the fluorescence (FL) spectra from the bulk diamond, the 156 nm and the 48 nm NDs. Each NV charge state is characterized by its fluorescence spectrum, with zero-phonon lines (ZPL) at 575 nm and 638 nm for the NV0 and NV-, respectively. In addition, a phonon sideband, peaked around 620 nm for the NV0 and 700 nm for the NV-, is observed, extending up to ≈ 850 nm in both cases. The FL spectra are normalized to the NV- ZPL for comparison. Moreover, NDs showed a larger component from the NV0s with respect to the bulk diamond (represented as black and light red curves, respectively). This is consistent with the idea that surface effects favor the NV0 centers [45,50], thus affecting the relative concentration of the different charge states.
Fig. 2 d,e,f show the ODMR spectra of the bulk diamond and NDs at different laser powers (from 1 to 30.5 mW) at constant MW power of 15 dBm. The ODMR spectra show the NV- central lines (AL and AR) and two side bands (BL and BR), the latter related to the hyperfine interaction between the NV- spins and the 13C nuclear spins . The 13C sidebands are separated from each other by ~130 MHz, consistently with previously reported values [35,51–54]. For all samples, the linewidth of the resonance bands decreases with laser power, in agreement with the line-narrowing effect described by Jensen et al. . Alongside with the increase in signal-to-noise (SNR) ratio, this phenomenon improves resolution of the NV- strain-split doublet (central dips AL and AR). We did not observe any detectable temperature effect on the position of the ODMR resonance that may be caused by absorption of MW or laser power .
In bulk diamond, a monotonic increase in ODMR contrast with laser power was observed, consistent with increasing NV- polarization levels [Fig. 2d and 3a]. Contrast was calculated as the mean value from the central resonances AL and AR. Interestingly, in NDs, a non-monotonic behavior was observed, as shown in Fig. 3 b,c. Up to 8.1 mW of laser power the ODMR contrast increases. At the highest power levels, the trend is the opposite, with a decrease in contrast systematically observed in different sample regions and for both ND sizes [Fig.3 b,c].
In principle, a reduction of the ODMR contrast in NDs observed at the highest laser power might result from the competition between optical and MW irradiation. We note that all experiments were performed at constant MW power, while laser power was varied. As a result, at the highest laser power, the spin system may be optically repolarized, with a net decrease in the observed ODMR contrast. However, this does not appear to be the case in this set of experiments. Indeed, the opposite effect is observed in the native bulk material, which has the same composition and concentration of nitrogen as the NDs, and was studied under identical experimental conditions. Moreover, the laser power density in our experiments is very low (up to 3μW/mm2), and far from saturation at the MW power used. This is orders of magnitude smaller than laser power saturation levels previously reported [56,57].
A practical difficulty in assessing the properties of ensembles of NDs is the large variability of the optical response in heterogeneous samples [42,58–60]. This has been ascribed to inter-aggregate interactions, size distribution and different efficiency in NV center initialization in ND aggregates . To circumvent this problem, we resorted to use a wide-field fluorescence microscope to spatially resolve ODMR spectra in different parts of the sample. The heterogeneity in ND deposition is apparent in Fig.3 b,c, where the wide-field FL images clearly show inhomogeneous aggregation and concentration of NDs. Aggregation is particularly evident for the smallest NDs of 48 nm. ODMR contrast in these samples depends on the selected ROIs, characterized by different levels of FL (see caption of Fig. 3), with lower variability observed in the 156 nm NDs compared to the 48 nm NDs. Conversely, the ODMR contrast from the bulk diamond is uniform throughout the image. Despite region and sample dependent contrast, our data show consistent trends for NDs in different samples and ROIs, thus suggesting that the effect here reported is robust and reproducible.
Charge dynamics and spin properties of NVs also depend on the ND microenvironment. To explore the effects described above in a typical bioassay, we incubated the NDs in cell cultures of macrophages (RAW 264.7). Internalization in macrophages is described in the Methods and illustrated by the confocal images of Fig. 4. The composite panel on the right shows the NDs (orange) internalized in the cells, together with the actine filaments (green) and the nuclei (blue), for both 156 nm NDs (top row) and 48 nm NDs (bottom row). The images show that NDs accumulate in the cytoplasm without accessing the nuclei. The cells underwent a rinsing procedure to remove most of the non-internalized NDs that could otherwise contribute with a confounding background emission. Hence, the signals reported in these images correspond to a large extent to internalized NDs.
The concentration of the 48 nm NDs in the native suspension appears to be too low to provide sufficient fluorescence signal after incubation with cells to measure spatially resolved ODMR spectra. Hence, in the following, we focus only on the 156 nm NDs.
In Fig. 5a we show the fluorescence images using ROIs of different size and location in the cell culture incubated with the 156nm NDs. Representative examples of ODMRs curves extracted from single-cell ROIs (~6x6 µm) or cell-aggregates ROI (from ~40x40 µm to ~100x100 µm) are shown in Fig. 5b and Fig. 5c, respectively. Despite some line broadening, compared to the bare NDs, the NV- central bands and 13C sidebands can still be resolved. As in the case of bare NDs, the linewidth of the resonances decreases with increasing laser power, while the SNR increases, therefore improving the resolution of the NV- doublet. Also in this case, we do not observe any variation depending on temperature (i.e., no shift of central resonances), indicating negligible heating by MW absorption by water in the cells or in the biological environment.
Fig. 5d shows the evolution of ODMR contrast with laser power for different ROIs. Interestingly, the ODMR contrast behavior at high laser power is more variable than in the bare NDs of Fig. 3b,c, ranging from a small reduction, to a plateau-like behavior or even a slight increase. We speculate that this wider heterogeneity may be due to differences in the microenvironment, and particularly to the different pH of various cellular compartments (e.g., pH ≈ 5 in lysosomes compared to pH ≈ 7.2 in the cytoplasm).
Indeed, pH can affect the functional groups at the ND oxidized surface (carboxylic acids, ketones, alcohols, esters, etc.), thus changing the properties and charge stability of shallow NV centers [61,62]. At low pH, e.g., carboxylates will be protonated to a much higher extent than under physiological conditions at pH ≈ 7.2, with potential effects on charge state of nearby NV centers. While plausible, this hypothesis requires further investigation.
As a faster method to assess the heterogeneity of the ODMR spectra in cells (Fig. 5 e,f), we set up a simple procedure. Specifically, we acquired two fluorescence images, with MW on- and off- resonance. Taking their difference and then normalizing the fluorescence signals pixelwise, it is possible to reconstruct an ODMR contrast image (Fig. 5g). In these ODMR contrast maps, an average ≈ 4.5% contrast is observed. Moreover, parts with a high (red) and a low (blue) ODMR contrast can be resolved within the cell.
This technique, much faster that the acquisition of the full ODMR spectrum, could prove useful in fast mapping of ODMR, thus paving the way to real-time, spatially resolved imaging of temperature [8,63], magnetic fields [22,31,64] or paramagnetic centers in live-cell bioimaging assays [46,65]. We note that, being based on the ratio between FL levels and not on the absolute FL values, this technique is more affected by noise fluctuations. Here, we applied a 1% contrast threshold in the ODMR contrast mapping to minimize the contributions of undesired spurious signal.