Wide-field fluorescent nanodiamond spin measurements toward real-time large-area intracellular thermometry

At a Glance

Metadata	Details
Publication Date	2021-02-19
Journal	Scientific Reports
Authors	Yushi Nishimura, Keisuke Oshimi, Yumi Umehara, Yuka Kumon, Kazu Miyaji
Institutions	Osaka City University, Keio University
Citations	47
Analysis	Full AI Review Included

Technical Documentation & Analysis: Wide-Field NV-Diamond Thermometry

Reference: Nishimura et al., Wide-field fluorescent nanodiamond spin measurements toward real-time large-area intracellular thermometry, Scientific Reports (2021) 11:4248.

Executive Summary

This research validates the feasibility of using camera-based wide-field Optically Detected Magnetic Resonance (ODMR) of Nitrogen Vacancy (NV) centers in nanodiamonds (NDs) for real-time, large-area intracellular thermometry, presenting a critical step toward advanced quantum sensing in biological systems.

Core Achievement: Demonstrated that wide-field ODMR detection provides temperature sensitivity comparable to traditional, point-by-point confocal detection (1.7-2.2 K/√Hz in living cells).
Methodology: Compared EMCCD camera-based wide-field detection against Avalanche Photodiode (APD) confocal detection using a home-built microscope and antenna-integrated culture dishes.
Artifact Mitigation: Identified and provided solutions for key wide-field artifacts, including background fluorescence, pixel saturation (Full-Well Capacity, FWC), and positional drift of NDs within the cell.
Biological Application: Successfully measured temperature shifts (e.g., -2.8 K) in living HeLa cells by monitoring the temperature-dependent shift of the NV center zero-field splitting frequency.
Future Integration: The findings establish the necessary technical parameters for integrating rapid, multi-point ODMR protocols essential for achieving high-precision, real-time thermal imaging across large biological fields of view.
Material Requirement: The study highlights the need for high-quality diamond materials and precise integration techniques (microwave antennas, optical coupling) to maximize signal-to-noise ratio (SNR) and sensitivity.

Technical Specifications

The following hard data points were extracted from the experimental results and setup description:

Parameter	Value	Unit	Context
NV Center Resonance Frequency (D₀)	~2.87	GHz	Zero-field splitting frequency
Excitation Wavelength	532	nm	Continuous-Wave (CW) Laser
Microwave Frequency Sweep Range	2.810 to 2.93	GHz	ODMR measurement range
Microwave Frequency Step (Δf_step)	1	MHz	Frequency sweep resolution
ND Particle Size (Nominal)	100	nm	NDNV100nmHi10ml
NV Centers per ND Particle	~500	-	Estimated ensemble size
Confocal Integration Time (At_pc)	100	ms	Per frequency point
Wide-Field Exposure Time (At_exp)	10	ms	Per image frame
EMCCD Full-Well Capacity (FWC)	185,000	e^-	Single pixel saturation limit
EMCCD Digital Resolution	16	bit	Analog-to-Digital Converter (ADC)
Temperature Dependence (dD/dT)	-74	kHz/K	Used for sensitivity calculation
Wide-Field Sensitivity (In Cells)	1.7 to 2.2	K/√Hz	Achieved in living HeLa cells
Detected Temperature Change (HeLa)	-2.8	K	Shifted center frequency by 210 kHz
Incubator Temperature Stability	±0.25	K	Measured over 250 min
Microwave Magnetic Field (B_MW)	2-5	Gauss	Estimated in 20 µm from antenna

Key Methodologies

The experiment utilized a hybrid optical-microwave setup to perform comparative ODMR measurements in both confocal and wide-field modes on nanodiamonds (NDs) internalized by HeLa cells.

Cell and ND Preparation: HeLa cells were incubated with 10 µg/mL NDs (NDNV100nmHi10ml) in collagen-coated, antenna-integrated glass-based dishes. Phenol red was removed from the transduction medium to prevent heat generation from the 532 nm excitation laser.
Optical Setup: A home-built microscope used a 532 nm CW laser for excitation via an NA 1.4 oil-immersion objective. Fluorescence was collected through a dichroic beam splitter and long-pass filter.
Microwave Delivery: Microwaves (2.810-2.93 GHz) were generated, amplified (45 dB), and delivered via a 25 µm copper wire linear antenna integrated into the culture dish.
Confocal Detection: Fluorescence was coupled to an optical fiber (acting as a pinhole) and detected by a gated Avalanche Photodiode (APD). The APD was gated (200 µs width) for microwave ON and OFF states.
Wide-Field Detection: The excitation laser was focused on the back focal plane to illuminate the entire field of view. Fluorescence was imaged onto an Electron Multiplying Charge-Coupled Device (EMCCD) camera (16-bit resolution, 10 ms exposure time, Gain G=10).
ODMR Spectrum Generation: ODMR contrast was calculated by dividing pixel values of microwave ON images (signal) by microwave OFF images (reference). Spectra were fitted with Gaussian functions to determine the center frequency shift (Δω), which is proportional to temperature change.
Temperature Control: Dish temperature (T_d) was precisely controlled using a PID-feedback system regulating foil heaters wrapped around the objective and metal cap, calibrated using a high-precision Pt100 thermistor.

6CCVD Solutions & Capabilities

This research demonstrates the potential of NV-diamond quantum sensing for high-throughput biological applications. Replicating and scaling this technology requires high-quality, customized diamond materials and precise integration capabilities—core competencies of 6CCVD.

Applicable Materials

To advance from nanodiamond ensembles to integrated, high-coherence quantum sensors suitable for real-time, large-area imaging, 6CCVD recommends the following MPCVD diamond materials:

Research Requirement	6CCVD Material Recommendation	Rationale
High-Coherence NV Centers	Optical Grade Single Crystal Diamond (SCD)	SCD offers superior crystal quality, minimal strain, and high purity, which are essential for maximizing NV center coherence time (T₂) and achieving the highest possible temperature precision (η_T).
Large-Area Sensor Arrays	Optical Grade Polycrystalline Diamond (PCD)	For scaling up to large-area imaging platforms (as targeted by wide-field detection), 6CCVD provides PCD plates up to 125 mm in diameter, enabling the fabrication of inch-scale sensor chips.
Integrated Quantum Devices	Custom Thin SCD/PCD Layers	The study requires thin layers for efficient NV detection. 6CCVD supplies SCD and PCD layers ranging from 0.1 µm to 500 µm thickness, ideal for creating membranes, waveguides, or shallow NV implantation substrates.
Electrochemical Sensing (Extension)	Boron-Doped Diamond (BDD)	While not the focus of this paper, BDD is available for researchers extending NV thermometry to include simultaneous electrochemical measurements in biological environments.

Customization Potential

The integration of wide-field ODMR into a functional biological microscope requires highly customized diamond components, particularly concerning microwave delivery and optical coupling. 6CCVD is uniquely positioned to meet these engineering demands:

Custom Dimensions and Shaping: 6CCVD provides plates and wafers up to 125 mm (PCD) and offers precise laser cutting and shaping services to create custom geometries required for antenna-integrated culture dishes or integrated diamond chips.
Advanced Metalization: The experiment relies on precise microwave antenna integration. 6CCVD offers in-house metalization capabilities including Au, Pt, Pd, Ti, W, and Cu deposition, allowing researchers to design and implement optimized microwave circuitry (e.g., Ti/Pt/Au stacks) directly onto the diamond substrate.
Ultra-Low Roughness Polishing: To minimize light scattering, background fluorescence, and maximize photon collection efficiency (critical for wide-field SNR), 6CCVD guarantees superior surface quality: Ra < 1 nm for SCD and Ra < 5 nm for inch-size PCD.

Engineering Support

The authors noted that achieving high precision requires careful parameter optimization, particularly regarding camera selection (higher FWC/bit depth) and managing artifacts like pixel saturation.

6CCVD’s in-house PhD team specializes in the material science and engineering of MPCVD diamond for quantum applications. We can assist researchers in similar NV-Diamond Thermometry projects by providing expert consultation on:

Selecting the optimal diamond grade (SCD vs. PCD) based on required NV coherence and sensor area.
Designing substrate dimensions and metalization layouts for efficient microwave coupling and optical integration.
Optimizing diamond surface preparation to ensure compatibility with biological cell culture and minimize optical loss.

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/s41598-021-83285-y