Simultaneous label-free live imaging of cell nucleus and luminescent nanodiamonds

At a Glance

Metadata	Details
Publication Date	2020-06-17
Journal	Scientific Reports
Authors	Michal Gulka, Hamideh Salehi, Béla Varga, Elodie Middendorp, Orsolya Páll
Institutions	Laboratoire Charles Coulomb, Laboratoire Bioingénierie et Nanosciences
Citations	15
Analysis	Full AI Review Included

Technical Documentation & Analysis: Simultaneous Label-Free Live Imaging using Fluorescent Nanodiamonds

This document analyzes the research paper “Simultaneous label-free live imaging of cell nucleus and luminescent nanodiamonds” (Scientific Reports, 2020) and outlines how 6CCVD’s advanced Chemical Vapor Deposition (CVD) diamond materials and processing capabilities can support, replicate, and extend this critical work in intracellular quantum sensing and bio-imaging.

Executive Summary

This research introduces a robust, label-free methodology combining Confocal Photoluminescence (PL) and Raman imaging to simultaneously visualize the cell nucleus and localize fluorescent nanodiamonds (fNDs) containing Nitrogen-Vacancy (NV) centers.

Core Achievement: Demonstrated simultaneous, label-free visualization of the cell nucleus and precise localization of fND probes within the diffraction-limited volume of the confocal microscope.
Label-Free Nucleus Imaging: Achieved high-contrast nucleus visualization by mapping the intensity and shape of the C-H stretching mode Raman peak (2800-3010 cm^-1), eliminating the need for fluorescent dyes or cell fixation.
Quantum Sensing Compatibility: The methodology is fully compatible with NV-based quantum sensing measurements in living cells, enabling time-dependent intracellular studies (e.g., relaxometry sensing).
Sensitive Localization: K-means cluster analysis (KMCA) of combined spectral data successfully distinguished internalized fNDs from the cellular environment, enhancing detection sensitivity by more than six times compared to standard fingerprint region analysis.
Material Robustness: The technique was verified across three distinct cell lines (MCF7, 184A1, DPSC) and proven effective on both living and fixed cell samples.
Extensibility: The method is applicable in principle to any red- and near-infrared-luminescent quantum probes, including Silicon-Vacancy (SiV) and Germanium-Vacancy (GeV) centers.

Technical Specifications

The following hard data points were extracted from the experimental methodology:

Parameter	Value	Unit	Context
Nanodiamond Source Material	HPHT	N/A	Used for fND production (5-50 nm size range)
Electron Irradiation Energy	16.6	MeV	Used to create vacancies for NV center formation
Electron Irradiation Dose	8.11 x 10¹⁸	particles cm^-2	Applied to the fND material
Annealing Temperature	900	°C	1 hour annealing post-irradiation
Oxidation Temperature	510	°C	6 hours oxidation in air
Excitation Wavelength	532	nm	Frequency-doubled Nd:YAG laser
Laser Power at Objective	20	mW	Used for Raman/PL acquisition
C-H Stretching Mode Peak	2915	cm^-1	Primary Raman peak used for nucleus visualization
NV Emission Region	670 - 890	nm	Region where ~70% of NV luminescence is detected
Lateral Resolution (f_lateral)	325	nm	Diffraction-limited resolution of the confocal system
Axial Resolution (r_axial)	991	nm	Diffraction-limited resolution of the confocal system
Cell Substrate	CaF₂	N/A	Used for cell growth and focusing aid

Key Methodologies

The experiment relied on precise material preparation and advanced spectral analysis to achieve simultaneous imaging:

Nanodiamond Preparation: HPHT nanodiamonds (5-50 nm) were purified, oxidized, and subjected to 16.6 MeV electron irradiation.
NV Center Formation: The irradiated material was annealed at 900 °C for 1 hour and subsequently oxidized at 510 °C for 6 hours to stabilize the NV centers and surface chemistry.
Cell Incubation: MCF7, 184A1, and DPSC cells were incubated with fND solution (30 µg/ml) for 1 hour. Samples were rinsed and kept in DPBS for live-cell Raman measurements.
Confocal PL/Raman Acquisition: Spectra were collected in a single scan using a Witec Confocal Raman Microscope (532 nm excitation, 20 mW power at objective).
Nucleus Visualization (Label-Free): The cell nucleus was visualized by mapping the integrated intensity of the C-H stretching mode (2800-3010 cm^-1), exploiting the protein/lipid ratio contrast.
fND Localization (KMCA): K-means cluster analysis (KMCA) was applied to the combined spectral data to identify and cluster pixels exhibiting NV luminescence, thereby localizing the fNDs inside or outside the cell.

6CCVD Solutions & Capabilities

6CCVD provides the high-quality, customizable CVD diamond materials necessary to advance research in quantum sensing, bio-imaging, and drug delivery applications demonstrated in this paper.

Applicable Materials for Quantum Bio-Sensing

While the paper utilized HPHT nanodiamonds, the next generation of high-performance quantum sensors requires the superior purity and control offered by MPCVD diamond.

Research Requirement	6CCVD Material Solution	Technical Advantage
High-Coherence NV Centers	High-Purity Single Crystal Diamond (SCD)	SCD wafers with ultra-low native nitrogen (< 1 ppb) provide the ideal matrix for creating NV centers with maximized T₂ coherence times, essential for high-sensitivity quantum sensing (e.g., relaxometry).
Near-Infrared Probes (SiV/GeV)	Custom-Doped SCD/PCD	6CCVD offers controlled doping of Silicon or Germanium during growth, yielding stable, bright SiV (738 nm) or GeV (637 nm) centers. These probes align perfectly with the paper’s goal of extending the method to red/near-infrared luminescent probes.
Scalable Nanodiamond Production	Polycrystalline Diamond (PCD) Plates	PCD wafers (up to 125 mm diameter) provide a cost-effective, large-area source material for high-throughput production of fNDs via subsequent milling and irradiation processes.
Boron-Doped Diamond (BDD)	BDD Substrates	For electrochemical sensing or integration into microfluidic systems requiring conductive diamond electrodes, BDD offers robust, chemically inert surfaces compatible with bio-environments.

Customization Potential

6CCVD’s in-house engineering and fabrication services directly address the specialized needs of quantum bio-sensing integration:

Custom Dimensions: We supply SCD and PCD plates in custom thicknesses (0.1 µm to 500 µm) and large formats (PCD up to 125 mm), providing optimal bulk material for subsequent electron irradiation and milling into fNDs.
Advanced Polishing: We offer ultra-smooth polishing (Ra < 1 nm for SCD, Ra < 5 nm for inch-size PCD). Low surface roughness is critical for minimizing strain and defects in the near-surface region of the diamond, which is vital for high-quality NV center creation and surface functionalization.
Metalization Services: For integrating diamond substrates into complex quantum measurement setups (e.g., combining PL/Raman with magnetic resonance detection), 6CCVD provides custom metalization layers (Au, Pt, Pd, Ti, W, Cu) for robust electrical contacts and device integration.

Engineering Support

6CCVD’s in-house PhD team specializes in the material science of CVD diamond for quantum applications. We offer consultation services to assist researchers in:

Material Selection: Optimizing diamond purity and doping levels (N, Si, Ge) to maximize the performance (brightness, coherence time) of color centers for intracellular quantum sensing projects.
Post-Processing Guidance: Advising on optimal substrate thickness and surface preparation necessary for efficient electron irradiation and subsequent milling into high-quality fNDs.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

Tech Support

Original Source

DOI: https://doi.org/10.1038/s41598-020-66593-7