Nanodiamonds as multi-purpose labels for microscopy
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2017-04-03 |
| Journal | Scientific Reports |
| Authors | Simon R. Hemelaar, Pascal de Boer, Mayeul Chipaux, Wilco Zuidema, Thamir Hamoh |
| Institutions | Delft University of Technology, University Medical Center Groningen |
| Citations | 94 |
| Analysis | Full AI Review Included |
Technical Analysis: Nanodiamonds as Multi-Purpose Labels for Advanced Microscopy
Section titled âTechnical Analysis: Nanodiamonds as Multi-Purpose Labels for Advanced MicroscopyâThis document provides a technical analysis of the research paper âNanodiamonds as multi-purpose labels for microscopyâ for 6CCVD, specialists in high-quality MPCVD diamond materials. The analysis focuses on validating the use of Nitrogen-Vacancy (NV) center-rich Fluorescent Nanodiamonds (FNDs) as robust, multimodal probes and aligning the material requirements with 6CCVDâs core capabilities.
Executive Summary
Section titled âExecutive SummaryâThis research paper firmly establishes sub-100 nm fluorescent nanodiamonds (FNDs) containing NV centers as the premier platform for multi-parameter correlative microscopy, overcoming major limitations inherent in organic and biological fluorophores.
- Multimodality Confirmed: FNDs were successfully used simultaneously for fluorescence imaging (FM), high-resolution structural imaging (Secondary/Backscattered Electron Microscopy - SE/BSE), cathodoluminescence (CL), and optically detected magnetic resonance (ODMR).
- Nanoscale Targeting & Uptake: Direct antibody-targeted labeling was achieved using FNDs as small as 70 nm, while 40 nm FNDs demonstrated successful cellular internalization by macrophages.
- Unprecedented Stability: FND fluorescence proved highly resistant to photobleaching and survived the rigorous sample preparation required for Electron Microscopy (EM), including osmium tetroxide fixation and Epon embedding, a critical bottleneck in Correlative Light and Electron Microscopy (CLEM).
- High-Resolution Localization: The intrinsic high Secondary Electron (SE) yield and stable CL properties of FNDs enable high-resolution localization within EM sections, moving beyond the diffraction limits of conventional light microscopy.
- Quantum Sensing Integration: Magnetic sensing using ODMR was successfully demonstrated on internalized FNDs (40 nm), paving the way for in situ nanoscale temperature, magnetic, or electric field measurements correlated directly with ultrastructure.
- Material Validation: The demonstrated resilience and quantum properties validate high-purity Single Crystal Diamond (SCD) as the essential base material for next-generation quantum sensing and bio-imaging probes.
Technical Specifications
Section titled âTechnical SpecificationsâThe following table summarizes the critical material and process parameters established by the research:
| Parameter | Value | Unit | Context |
|---|---|---|---|
| Nanodiamond Size (FND40 Average) | 54 ± 26 | nm | Derived from SE imaging analysis (1141 particles) |
| Nanodiamond Size (FND70 Average) | 67 ± 37 | nm | Derived from SE imaging analysis (5051 particles) |
| NV Center Count (FND40) | 10-15 | centers | Specified by supplier |
| NV Center Count (FND70) | >300 | centers | Specified by supplier |
| EM Section Thickness | 300 | nm | Prepared using ultramicrotome for CLEM/CL |
| Electron Beam Excitation (CL/SE) | 3 | keV | Optimized low energy for CL detection |
| Electron Beam Current (CL/SE) | 0.8 | nA | Used at 7 mm working distance |
| EM Imaging Voltage (BSE) | 10 | kV | Used for high vacuum backscatter detection |
| ODMR Resonance Frequency | 2.87 | GHz | Characteristic NV- zero-field splitting |
| ODMR Contrast (FND40, internalized) | 3.3 | % | Contrast between resonance line and background |
| ODMR Acquisition Time | 13 | min | Averaging 300 individual runs |
| Fluorescence Emission Peak | ~660 | nm | Observed peak intensity in the red spectrum |
Key Methodologies
Section titled âKey MethodologiesâThe experiment successfully combined bio-conjugation, cell culturing, harsh fixation, and advanced correlative imaging techniques.
- Nanodiamond Characterization:
- FND40 (40 nm average) and FND70 (70 nm average) powders were drop-casted onto ITO-coated cover glasses.
- FND size and dispersity were analyzed using Secondary Electron (SE) detection on a FEI Verios SEM.
- Cell Preparation & Internalization (FND40):
- J774A.1 macrophages were incubated with 1 ”g/ml FND40 for 5 hours at 37 °C / 5% CO2.
- Cells were fixed using 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M cacodylate buffer.
- Immunolabeling (FND70):
- HT29-EpCAM-GFP cells were labeled using a three-step process: anti-EpCAM monoclonal antibody, biotinylated rabbit anti-mouse IgG linker, and finally, streptavidin-conjugated FND70 (1:20 weight ratio).
- Correlative Sample Embedding:
- Fixed cells (J774 and HT29) underwent conventional EM preparation, including post-fixation with 1% osmium tetroxide/1.5% potassium ferrocyanide.
- Cells were dehydrated through graded ethanol series and embedded overnight in Epon at 58 °C.
- Epon blocks were sectioned at 300 nm thickness using an ultramicrotome onto ITO-coated glass slides.
- Multi-Modal Imaging (CLEM/CL/ODMR):
- Fluorescence Preservation Check: Fluorescence preservation after embedding was confirmed using a Zeiss LSM780 confocal microscope.
- Integrated EM/CL: Sections were imaged using a SECOM integrated microscope on a Zeiss Supra55 SEM at high vacuum. CL was recorded simultaneously via a PMT detector, and SE/BSE images were captured for ultrastructural correlation.
- Quantum Sensing (ODMR): A home-built confocal diamond magnetometer was used. Microwave excitation (2.87 GHz) was delivered via a microwire antenna, and the resulting change in fluorescence intensity was measured using an Avalanche Photodiode.
6CCVD Solutions & Capabilities
Section titled â6CCVD Solutions & CapabilitiesâThe findings confirm that NV-FNDs are the ideal material for combining quantum sensing (ODMR) with structural biology (CLEM/CL/EM). 6CCVD provides the necessary high-quality MPCVD diamond substrates and customized engineering services required to replicate and scale this cutting-edge research.
Applicable Materials for Replication and Scaling
Section titled âApplicable Materials for Replication and ScalingâThe core requirement of this research is a stable, high concentration of NV centers. This is achieved through specific nitrogen incorporation during growth and subsequent high-temperature annealing of high-purity Single Crystal Diamond (SCD).
| 6CCVD Material | Application in Research Context | Specific Capability Link |
|---|---|---|
| Optical Grade SCD (0.1”m - 500”m) | NV Center Precursor: The highest purity base material required for generating FNDs with reproducible, high-contrast NV or SiV centers (essential for ODMR/CL). | SCD wafers up to 125mm for large-scale FND production and superior crystalline consistency. |
| Polycrystalline Diamond (PCD) Wafers | High-Volume Precursor: For bulk production of FNDs where ultra-high quantum coherence is not strictly necessary, PCD offers scalable, cost-effective diamond material. | PCD wafers up to 125mm, enabling large surface area processing for industrial ND derivation. |
| Boron-Doped Diamond (BDD) Substrates | Conductive Substrates: For next-generation EM/CL imaging, BDD wafers provide intrinsically conductive substrates, eliminating the need for complex ITO coating or thin film metalization used in the paper. | Customizable BDD substrates (up to 10mm thickness) capable of acting as electrode platforms for integrated bio-electronic devices. |
Customization Potential for Advanced Quantum Probes
Section titled âCustomization Potential for Advanced Quantum ProbesâTo move from proof-of-principle work to commercial application, researchers require tailored dimensions, surface functionalization, and integrated substrate engineering.
- Custom Dimensions and Etching: 6CCVD routinely provides custom diamond plates (up to 125mm) which can be further processed via laser cutting services to create unique geometries or precisely defined starting materials for ND production, ensuring geometric consistency far exceeding simple mechanical crushing.
- Integrated Metalization and Substrate Engineering: The paper utilized ITO-coated glass for conductivity in the SEM/CL process. 6CCVD offers in-house deposition of standard and specialized metal layers directly onto the diamond:
- Au, Pt, Pd: Ideal for creating highly biocompatible, functionalized surfaces for streptavidin/antibody conjugation and subsequent signal stabilization.
- Ti, W, Cu: Essential for integrated microwave circuitry fabrication (as required for the ODMR antenna shown in Figure 1c), enabling scalable on-chip quantum sensing arrays.
- Polishing Standards: 6CCVD guarantees ultra-low roughness (Ra < 1nm for SCD, Ra < 5nm for inch-size PCD), which is crucial for maximizing surface area control and ensuring uniform surface chemistry during the functionalization and bio-conjugation steps (e.g., precise streptavidin attachment).
Engineering Support & Conclusion
Section titled âEngineering Support & ConclusionâThis research highlights the necessity of precise material engineering at both the bulk (SCD plate) and nanoscale (FND) level. 6CCVDâs in-house PhD team can assist with material selection for similar Correlative Microscopy and Nanoscale Quantum Sensing projects, ensuring optimal NV center density, crystal orientation, and substrate preparation for integration into complex electron beam or ODMR systems.
For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.