I. Introduction: The Fundamental Challenges in Microenvironment Research in the Life Sciences

Reactive oxygen species and free radicals (ROS, such as the superoxide anion •O₂⁻ and the hydroxyl radical •OH) are central signaling molecules in cellular metabolism, immune regulation, inflammatory responses, and the evolution of the tumor microenvironment. Taking macrophages as an example, during their processes of pathogen phagocytosis, clearance of cellular debris, and modulation of inflammation, intracellular vesicles—namely endosomes/phagosomes—undergo a burst‑like production of large quantities of free radicals, which serve to mediate antimicrobial activity and signal transduction.

However, for a long time, the life sciences have consistently lacked a method capable of… Non‑invasive, long‑term dynamic monitoring at the cellular and subcellular scales. A low‑disturbance detection method. How to acquire in situ, high spatiotemporal‑resolution data on endogenous molecular fluxes remains a longstanding technical bottleneck for elucidating cellular stress mechanisms and immune homeostasis.

II. Methodological Limitations of Traditional Detection Methods

At present, the biomedical community primarily relies on small-molecule fluorescent dyes or genetically encoded probes to assess intracellular free radicals. However, conventional approaches suffer from intractable methodological limitations when applied to long-term studies of complex biological systems:

Biochemical Consumption and Physiological Interference: Traditional probes typically rely on redox reactions with free radicals to produce color, which means that… The act of measurement itself continuously consumes the target molecule. , thereby artificially disrupting the cells’ intrinsic redox balance and native physiological processes.

Photobleaching and Phototoxicity: Continuous high‑frequency excitation light irradiation causes rapid photobleaching of the dye, and its photochemical byproducts exhibit significant cytotoxicity, thereby severely limiting the feasibility of long‑term, continuous dynamic monitoring.

Multidimensional Comparison of Core ROS Detection Methodologies

For the detection of complex intracellular microenvironments, existing mainstream technological approaches each have their own limitations:

III. The Fundamental Physical Principle of NV Center Quantum Sensing

To overcome the fundamental limitations of chemical probes, this study introduces solid-state spin quantum sensors. At its core, the underlying physical principle leverages the intrinsic properties of materials, enabling a paradigm shift from “chemical‑reaction detection” to “physical‑field sensing”:

Spin Microenvironment Sensing Mechanism: Free radical molecules with unpaired electrons are, by nature, microscopic sources of magnetic noise. Meanwhile, within solid-state nanodiamonds (FNDs)… The nitrogen–vacancy (NV) center is a solid-state spin quantum probe that is extremely sensitive to local magnetic noise fluctuations. 。

Lossless T1 Relaxation Modulation: When diamond nanoparticles are internalized into vesicles via cellular endocytosis, fluctuations in the local free-radical concentration give rise to pronounced variations in paramagnetic noise, which directly modulate the spin‑lattice relaxation time (T1) of NV centers. By means of nondestructive optical readout, this spin‑physical signal can be precisely retrieved, enabling nondestructive quantitative characterization of the kinetics of local free-radical generation. The entire sensing process relies on purely physical interactions. Due to the exceptional chemical inertness of NV‑FNDs, they do not consume the target free radicals during measurement, thereby minimizing perturbation of native biological processes. 。

IV. Core Methodological Breakthroughs and Experimental Validation of the Paper

The research team successfully completed end-to-end validation within a highly complex primary macrophage system, achieving methodological rigor in experimental design. Nanoscale spatial positioning "and" Immunophenotypic Analysis “Cross-disciplinary integration:”

1. Local microenvironment sensing at the subcellular “intracellular vesicle scale”

By leveraging the intrinsic endocytic uptake of surface‑carboxylated NV‑FNDs, the quantum probes are precisely localized within the endosomal vesicles inside macrophages. Their detection radius has been successfully reduced to Sub-50-nanometer scale , it has surpassed the diffraction limit of conventional optical imaging, enabling the acquisition of localized dynamic information at the subcellular resolution level.

2. Dynamic Analysis of the Immune Stress Flow in “Macrophage Polarization”

The experiment established a well-defined immunophenotypic control system through standard cytokine induction, enabling the assessment of differences in the kinetics of intracellular vesicular reactive oxygen species production under distinct polarization states.

3. Characterization of Key Experimental Results

Quantum Classification of Functional States: The experimental results show that, following stimulation with lipopolysaccharide (LPS), The T1 relaxation time within M1 macrophages is significantly shortened. , quantitatively confirming that the intravesicular free-radical generation rate and peak levels are significantly higher than those in M0 and M2 macrophages. This demonstrates that the NV-FNDs platform not only enables precise quantitative characterization of ROS‑generation kinetics but also accurately maps the functional phenotypes of immune cells under different stress conditions.

Spatiotemporal Tracking of Exogenous Oxidative Stress: In a series of several-hour-long drug‑induced stimulation experiments, the system continuously and seamlessly recorded the complete time‑resolved kinetic profile of exogenous hydrogen peroxide (H₂O₂) as it traversed the cell membrane from the extracellular space into the cytoplasm, triggering the generation of free radicals within vesicles and their subsequent dissipation.

Benchmarking next-generation performance:

V. The Upgrading and Prospects of Academic Paradigms

The significance of this work lies not merely in “detecting a particular free radical,” but also in establishing… “ NV‑center nanodiamond”   →   Intracellular vesicle localization   →   Local Magnetic Noise Sensing   →   Dynamic Analysis of Free Radicals the complete technological roadmap. Its true value lies in systematically introducing quantum sensing into the study of complex biological microenvironments for the first time.

By translating quantum-mechanical measurements (T1 relaxation modulation) into a standardized live-cell assay. , this study has established a completely new In-situ quantum sensing paradigm This not only lays a solid methodological foundation for subsequently extending this technology to three-dimensional organ-on-a-chip systems and the continuous dissection of the pathological microenvironment in complex human primary tissue sections, but also paves the way for acquiring—within the AI for Science era— High-fidelity, native lossless, multi‑scale spatiotemporal dynamic biological big data It provides an irreplaceable hardware-level path.

Image and Text Guide

Figure 1. Schematic diagram of macrophage polarization and the experimental design for treatment with fluorescent nanodiamonds (FND).

(a) An overview of the entire process by which macrophages internalize fluorescent nanodiamonds and subsequently polarize into M1 and M2 phenotypes. (b)   Macrophage polarization status was assessed and quantified using flow cytometry in combination with marker-based assays. (c)   Using confocal imaging and colocalization analysis, we examined the distribution and interactions of fluorescent nanodiamonds within macrophage endocytic vesicles, early endosomes (early endosome antigen‑1, light red), and late endosomes/lysosomes (lysosomal-associated membrane protein 1, light blue). (d)   The free radical concentration in polarized macrophages was assessed using longitudinal relaxation time (T1) relaxation measurements, enabling the characterization of the cells’ local microenvironment.

Figure 2. Flow cytometric analysis of activation markers for M1 (panels a and b) and M2 (panels c and d) macrophages in cells treated with non-fluorescent nanodiamonds, following stimulation for 4 h, 24 h, and 48 h. The figure shows the median fluorescence intensity (MFI) for each group. Red asterisks indicate statistically significant differences compared with the M0‑type macrophage group cultured under the same conditions; only those comparisons that are statistically significant relative to the M0 group are labeled.

Figure 3. Flow cytometric analysis of M1‑type (panels a and b) and M2‑type (panels c and d) activation markers in cells treated with 70‑nm fluorescent nanodiamonds, at 4 h, 24 h, and 48 h post‑stimulation, to assess the effects of fluorescent nanodiamonds on macrophage polarization and marker expression. The figure shows the median fluorescence intensity (MFI) for each group. Red asterisks indicate statistically significant differences compared with the M0‑type macrophage group cultured under the same conditions.

Figure 4. Distribution of 70-nm fluorescent nanodiamonds (red) and early endosomes labeled with early endosome antigen 1 (EEA1, green) within macrophages exhibiting M0, M1, and M2 phenotypes at different time points. : Fluorescent nanodiamonds, treated and polarized for 30 min (before polarization), 4 h, and 24 h. Scale bar: 20 μm.

Figure 5. Distribution of 70-nm fluorescent nanodiamonds (red) and late endosomes/lysosomes labeled with lysosomal-associated membrane protein 1 (LAMP1, green) in macrophages exhibiting M0, M1, and M2 phenotypes at different time points: Fluorescent nanodiamonds, treated and polarized, at 30 min (before polarization), 4 h, and 24 h. Scale bar: 20 μm.

Figure 6. Retention rate of FND in macrophages at different time points (a), Co-localization with early endosomes   (b), and its colocalization with late endosomes/lysosomes   (c), Before macrophage activation (30 minutes after FND treatment), and at 4 hours and 24 hours post-activation.

Figure 7. T1 values of pre-activated macrophages treated with fluorescent nanodiamonds (FNDs) for 30 minutes, as well as of M0-, M1-, and M2‑type activated macrophages after polarization for 4 hours and 24 hours. We measured three fluorescent nanodiamonds per cell, six cells per variant, and repeated the experiment six times.