Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a subcellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable “zooming-in” to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery.

💡 Research Summary

The authors address a critical need in modern cell biology for a single labeling probe that can be visualized both in optical microscopy and in electron microscopy (EM) without cytotoxicity or photobleaching. They propose fluorescent nanodiamonds (NDs) containing nitrogen‑vacancy (N‑V) centers as the multimodal label. N‑V centers emit bright, stable red fluorescence under green laser excitation, enabling high‑contrast imaging in confocal or wide‑field fluorescence microscopes. At the same time, the crystalline diamond lattice produces a characteristic diffraction pattern when illuminated by an electron beam, allowing rapid detection of individual NDs in EM diffraction mode.

To demonstrate the concept, the team functionalized 30–100 nm NDs with anti‑Nup153 antibodies, targeting the nuclear pore complex (NPC) on the nuclear envelope. After incubating cultured HeLa cells with the ND‑antibody conjugates, they confirmed uptake and NPC localization by fluorescence microscopy. The fluorescent signal remained stable for at least 48 h, indicating negligible photobleaching and confirming the biocompatibility of the particles.

In the EM workflow, the authors first scanned the entire cell in diffraction mode. Each ND generated a distinct set of diffraction spots (point‑line‑ring pattern) that could be automatically identified by a custom image‑analysis script. This step provided precise XY coordinates of every ND within seconds, effectively “mapping” the cell. The coordinates were then fed into a high‑resolution TEM/ STEM acquisition routine, which zoomed in on the identified NPCs. The resulting TEM images revealed the ultrastructure of the nuclear envelope and the NPC scaffold at sub‑nanometer resolution, directly correlated with the fluorescence data.

Key technical advantages highlighted in the paper include:

1. True multimodality – The same physical particle supplies both optical fluorescence and electron diffraction signals, eliminating registration errors that plague correlative light‑EM (CLEM) approaches that rely on separate fluorophores and electron‑dense tags.
2. Non‑toxicity and photostability – Diamond is chemically inert, does not generate reactive oxygen species, and its fluorescence does not bleach, allowing long‑term live‑cell tracking and repeated EM processing (fixation, dehydration, embedding) without loss of signal.
3. Surface functionalization flexibility – The ND surface can be modified with a wide range of ligands (antibodies, peptides, nucleic acids), making the platform adaptable to any intracellular target, from organelles to specific protein complexes.
4. Single‑particle sensitivity – Both fluorescence and diffraction detection work down to the single‑ND level, enabling quantitative studies of uptake efficiency, trafficking pathways, and stoichiometry of molecular interactions.
5. Additional sensing capabilities – The N‑V center is a quantum sensor of magnetic fields and temperature. In principle, the same ND used for imaging could simultaneously report nanoscale magnetic fluctuations or local heating, opening avenues for multimodal functional imaging.

The authors also discuss limitations. ND particles smaller than ~20 nm produce weak diffraction signals that can be missed in routine EM scans, requiring longer exposure or higher beam currents that may damage the specimen. Antibody orientation on the ND surface is not fully controlled, leading to variability in targeting efficiency. The current study focuses solely on NPC labeling; extending the method to other organelles will require optimization of ligand density and possibly multiplexed labeling strategies (e.g., using N‑V centers with different charge states for spectral separation).

Overall, the paper establishes a robust workflow that couples rapid EM diffraction mapping with high‑resolution TEM imaging, anchored by a single, biocompatible nanodiamond label. By demonstrating that a single ND can be tracked from live‑cell fluorescence imaging through to nanometer‑scale EM structural analysis, the authors provide a powerful tool for correlating molecular identity with ultrastructure. Potential applications span drug‑delivery tracking, investigation of protein complex assembly, and quantum‑sensing‑enabled functional studies within living cells. The approach is scalable, compatible with existing microscopy infrastructure, and poised to become a cornerstone technique for multimodal cellular imaging.