| Metadata | Details |
|---|
| Publication Date | 2020-06-17 |
| Journal | Scientific Reports |
| Authors | Michal Gulka, Hamideh Salehi, BĂ©la Varga, Elodie Middendorp, Orsolya PĂĄll |
| Institutions | Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry, Hasselt University |
| Citations | 15 |
| Analysis | Full AI Review Included |
This research introduces a robust, label-free methodology for the simultaneous visualization of the cell nucleus and fluorescent nanodiamond (fND) probes in living cells, crucial for advancing intracellular quantum sensing.
- Core Achievement: Demonstrated simultaneous detection of NV center photoluminescence (PL) and label-free Raman chemical imaging of the cell nucleus in a single confocal scan.
- Enhanced Sensitivity: Achieved high-sensitivity fND localization by shifting the Raman grating center to 680 nm, capturing the majority of NV PL emission (670-890 nm) and increasing detection sensitivity by more than six times compared to standard settings.
- Label-Free Nucleus Imaging: Nucleus visualization is achieved without staining or fixation by mapping the C-H stretching mode Raman peak (2800-3010 cm-1), exploiting the inherent contrast based on the protein/lipid ratio.
- Chemical Localization: K-means cluster analysis (KMCA) is applied to the combined spectral data to chemically distinguish and localize fNDs, confirming their internalization and colocalization with the nucleus within the diffraction-limited volume.
- Probe Compatibility: The technique is compatible with any red- or near-infrared-luminescent cell probes (e.g., silicon-vacancy, quantum dots) and is fully compatible with NV quantum sensing measurements.
- Robustness: The method was successfully validated on three diverse cell lines (MCF7, 184A1, and DPSC) in both living and fixed states.
| Parameter | Value | Unit | Context |
|---|
| fND Particle Size Range | 5 - 50 | nm | HPHT, oxidized, majority 20 nm |
| Electron Irradiation Energy | 16.6 | MeV | Used for NV center creation |
| Electron Irradiation Dose | 8.11 x 1018 | particles cm-2 | Used for NV center creation |
| Annealing Temperature | 900 | °C | Post-irradiation treatment (1 h) |
| Oxidation Temperature | 510 | °C | Surface purification (6 h) |
| Excitation Wavelength | 532 | nm | Confocal Raman/PL microscopy (Nd:YAG laser) |
| Laser Power (Objective) | 20 | mW | Used for imaging |
| Objective Numerical Aperture (NA) | 1.0 | - | 60x Water Immersion |
| Lateral Resolution (flateral) | 325 | nm | Diffraction-limited resolution |
| Axial Resolution (raxial) | 991 | nm | Diffraction-limited resolution |
| Optimized Grating Center Wavelength | 680 | nm | Maximizes NV PL detection sensitivity (>6x increase) |
| NV PL Emission Range (High Intensity) | 670 - 890 | nm | Captures ~70% of NV emission |
| C-H Stretching Mode Range | 2800 - 3010 | cm-1 | Used for label-free nucleus visualization |
| Nucleus Visualization Range (Negative Image) | 2800 - 2935 | cm-1 | Highlights lipid-poor regions (nucleus) |
- fND Synthesis and Activation: High-pressure high-temperature (HPHT) nanodiamonds (5-50 nm) were purified, oxidized, and subjected to 16.6 MeV electron beam irradiation (8.11 x 1018 particles cm-2). This was followed by annealing at 900 °C for 1 h and subsequent oxidation at 510 °C for 6 h to create and activate NV centers.
- Cell Culture and Incubation: MCF7, 184A1, and DPSC cells were grown on CaF2 substrates. Cells were incubated with the prepared fND solution (30 ”g/ml) for 1 hour at 37 °C and 5% CO2 to facilitate internalization.
- Confocal PL/Raman Acquisition: Measurements were performed using a Witec Confocal Raman Microscope Alpha System 300 R with a 532 nm excitation laser (20 mW at the objective) and a 60x water immersion objective (NA = 1.0).
- Spectral Optimization: The detection grating was centered at 680 nm. This shift sacrifices the traditional Raman âfingerprint regionâ (700-1700 cm-1) but maximizes the collection of NV PL, enabling sensitive fND detection in a single, rapid scan.
- Label-Free Nucleus Mapping: The cell nucleus was visualized by mapping the intensity of the C-H stretching mode (2800-3010 cm-1). Specifically, the ânegative imageâ was generated by mapping the lipid-rich region (2800-2935 cm-1), which appears dark in the lipid-poor nucleus, providing high contrast.
- Data Processing and Localization (KMCA): K-means cluster analysis (KMCA) was applied to the combined PL/Raman spectra. This unsupervised algorithm clustered pixels based on the presence and intensity of NV luminescence and the C-H Raman signal, allowing for the distinction of fNDs inside versus outside the cell.
- Image Merging: The final image was constructed as two stacked layers: the C-H Raman map (nucleus visualization) merged with the KMCA-identified NV-luminescent pixels (fND localization), demonstrating colocalization.
| Industry/Field | Application Area | Technical Relevance |
|---|
| Quantum Sensing & Metrology | Intracellular Nanoscale Sensing | Provides essential localization data for NV-based quantum sensors (e.g., temperature, magnetic fields) within specific organelles, enabling precise measurement context. |
| Nanomedicine & Drug Delivery | Monitoring Nanoparticle Transport | Enables label-free, real-time tracking of fND-based drug or gene carriers, verifying internalization and successful delivery to the cell nucleus or other targets. |
| Biomaterials & Biocompatibility | Cytotoxicity and Uptake Studies | Offers a robust, non-toxic method to assess the interaction and accumulation of nanoprobes (fNDs, quantum dots) with living cells and nuclei over extended periods. |
| Advanced Microscopy | Label-Free Chemical Imaging | Utilizes the C-H stretching mode for rapid, high-contrast chemical mapping of protein/lipid ratios in cells, serving as a robust alternative to traditional fluorescent nuclear stains. |
| Probe Development | Red/NIR Probe Validation | The methodology is broadly applicable to localize any red- or near-infrared-luminescent probes (e.g., silicon-vacancy, germanium-vacancy centers) relative to cellular structures. |