Fluorescent nanodiamonds as a robust temperature sensor inside a single cell

At a Glance

Metadata	Details
Publication Date	2018-01-01
Journal	Biophysics and Physicobiology
Authors	T. Sekiguchi, Shingo Sotoma, Yoshie Harada
Institutions	The University of Osaka, Quantum (Australia)
Citations	77
Analysis	Full AI Review Included

Technical Analysis: Robust Single-Cell Thermometry Using NV- Center Nanodiamonds

This document analyzes the research paper, “Fluorescent nanodiamonds as a robust temperature sensor inside a single cell,” focusing on the material requirements (Nitrogen-Vacancy center diamonds) and translating the findings into actionable solutions provided by 6CCVD, an expert in MPCVD diamond engineering.

Executive Summary

This study validates Fluorescent Nanodiamonds (FNDs) containing negatively-charged Nitrogen-Vacancy (NV^-) centers as the industry standard for robust, quantitative nanoscale thermometry inside biological systems.

Unprecedented Robustness: The thermosensing ability of NV^- FNDs was experimentally proven to be virtually unaffected by extreme environmental factors, including shifts in pH (3.0 to 11.0), high ion concentrations (3 M NaCl), viscosity (glycerol), surface chemistry, and organic solvents.
Absolute Temperature Measurement: A novel, two-step ODMR (Optically Detected Magnetic Resonance) protocol enabled the first absolute temperature measurement inside a single living cell using a single FND, overcoming calibration challenges.
High Precision: Temperature measurement accuracy was achieved at ±0.5°C, meeting the necessary precision (better than ±1°C) for validating thermogenesis in cellular biology.
Core Mechanism: Temperature is determined by monitoring the shift in the Zero-Field Splitting (D) of the NV^- ground state spin sublevels, an intrinsic property of the diamond lattice.
Key Finding: The absolute temperature inside a single HeLa cell (33.5°C) was measured to be significantly higher (1.5°C) than the surrounding culture medium (32.0°C), supporting localized thermogenesis.
6CCVD Relevance: The creation of stable, high-density NV centers requires ultra-pure Single Crystal Diamond (SCD) material and precise defect engineering protocols—6CCVD’s core expertise.

Technical Specifications

Hard data points extracted from the experimental measurements relating to temperature sensing and ODMR spectroscopy.

Parameter	Value	Unit	Context
Target Measurement Accuracy	< ±1.0	°C	General requirement for thermal inhomogeneity studies.
Achieved Accuracy (Single Cell)	±0.5	°C	Absolute temperature reading for a single FND.
Live Cell Absolute Temperature	33.5	°C	Measured temperature inside a live HeLa cell.
Culture Medium Temperature	32.0	°C	Temperature of the surrounding culture medium.
Temperature Dependence (ΔD/ΔT)	-54 to -78	kHz/°C	Range observed across various tested environmental factors (pH, viscosity, etc.).
Zero-Field Splitting (D)	2868.55	MHz	D value measured from FND in the live cell.
MW Frequency Sweep Range	2820 to 2920	MHz	Range used for obtaining the full ODMR spectrum.
NV Center Excitation Wavelength	532	nm	Continuous Nd:YAG laser used for initialization.
Nanodiamond Annealing Temperature	800	°C	Vacuum process used to form NV^- centers.
Nanodiamond Oxidation Temperature	450	°C	Air process used to remove graphitic surface carbon.

Key Methodologies

The core steps required for material synthesis, functionalization, and measurement setup used to achieve robust, absolute thermometry.

I. Material Synthesis and Preparation

Diamond Precursors: Nanodiamond particles were pre-treated by Het ion irradiation to introduce defects necessary for NV creation.
NV Center Formation: Particles underwent high-temperature vacuum annealing (800°C) to mobilize vacancies and nitrogen atoms, leading to the formation of NV centers.
Purification: Diamond surfaces were oxidized in air at 450°C for 2 hours to remove graphitic carbon, yielding Fluorescent Nanodiamonds (FNDs).
Functionalization: FNDs were surface-coated with polyethyleneimine (PEI) in Milli-Q water via sonication and repeated centrifugation/wash processes to facilitate cellular uptake.

II. Optically Detected Magnetic Resonance (ODMR) Setup

Microscope Platform: System configured on an inverted fluorescence microscope (Nikon Ti-E).
Excitation: Continuous Nd:YAG laser (532 nm) used to initialize and readout the NV spin state.
Signal Capture: Fluorescence captured by an oil immersion objective lens (×100, NA=1.49), passed through a long-wavelength filter, and imaged onto an EMCCD camera (Andor, iXon860).
Microwave (MW) Delivery: A two-turn copper coil (~1 mm diameter) was placed immediately above the sample, driven by an amplified MW generator (Agilent E8257D) sweeping the frequency from 2820 to 2920 MHz.

III. Absolute Temperature Measurement Protocol

Live Cell Measurement: ODMR spectrum and D value (e.g., 2868.55 MHz) of a single internalized FND were measured in a live HeLa cell cultured at a maintained temperature (e.g., 32.0°C).
Calibration: The cell was chemically fixed (70% ethanol). Crucially, the diamond’s structure is inert to this treatment.
Fixed Cell Calibration: The ODMR spectra of the identical FND in the fixed cell were measured at three controlled external temperatures (27.0, 32.0, and 37.0°C) to establish the linear D vs. T calibration line.
Absolute Calculation: The D value measured in the live cell was referenced against the fixed cell calibration line to determine the absolute temperature (33.5°C).

6CCVD Solutions & Capabilities

This research highlights the critical need for highly controlled, robust diamond materials. 6CCVD is uniquely positioned to supply the foundational materials and engineering services required to advance NV-based thermometry and quantum sensing.

Applicable Materials

To replicate or extend this research into bulk or integrated sensing platforms, high-quality, ultra-low defect diamond is essential for reproducible NV center generation.

Material Requirement	6CCVD Recommended Solution	Technical Justification
High Purity Precursors	Optical Grade Single Crystal Diamond (SCD)	Ultra-low native nitrogen content (Type IIa) ensures controlled NV formation and maximized coherence time necessary for precision ODMR.
Thermal Robustness Testing	Polycrystalline Diamond (PCD) Wafers (up to 125 mm)	Provides large area platforms for developing and testing scalable micro-coil arrays and integrated NV sensors under high-power conditions.
Advanced Sensing	Custom Defect-Engineered SCD	Material customized for specific nitrogen doping levels and optimized post-processing (annealing and irradiation) to maximize NV^- yield and stability, mirroring the paper’s material requirements.

Customization Potential for NV-Based Research

6CCVD’s specialized fabrication services directly address the complex engineering challenges associated with integrating NV-based sensors into functional devices.

Precision Defect Engineering: 6CCVD’s in-house capabilities include customized growth and post-growth processing (high-temperature annealing, potentially ion implantation consultation) to control the location, concentration, and charge state (NV^- vs. NV⁰) of sensing centers.
Custom Dimensions and Substrates: While this paper used nanodiamonds, 6CCVD supplies plates and wafers up to 125 mm in PCD and thick SCD substrates (up to 500 µm), enabling the scaling of thermal sensor arrays or quantum components.
Micro-Coil and Interconnect Fabrication: The experimental setup relied on a specialized MW coil delivery system. 6CCVD offers extensive custom metalization services (Ti/Pt/Au, Cu, W) critical for depositing on-chip transmission lines and microwave delivery structures required for integrated ODMR sensing.
Surface Preparation: For advanced optical sensing setups, 6CCVD provides state-of-the-art polishing, achieving Ra < 1 nm on SCD and Ra < 5 nm on inch-size PCD, ensuring minimal surface scattering losses essential for high NA objective coupling.

Engineering Support

The successful replication of this work relies on stringent control over material purity and post-processing protocols. 6CCVD’s in-house PhD team can assist researchers and technical engineers with material selection, custom fabrication specifications, and integration design for projects focusing on nanoscale thermometry, quantum sensing, and biophysics applications.

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

View Original Abstract

Thermometers play an important role to study the biological significance of temperature. Fluorescent nanodiamonds (FNDs) with negatively-charged nitrogen-vacancy centers, a novel type of fluorescence-based temperature sensor, have physicochemical inertness, low cytotoxicity, extremely stable fluorescence, and unique magneto-optical properties that allow us to measure the temperature at the nanoscale level inside single cells. Here, we demonstrate that the thermosensing ability of FNDs is hardly influenced by environmental factors, such as pH, ion concentration, viscosity, molecular interaction, and organic solvent. This robustness renders FNDs reliable thermometers even under complex biological cellular environment. Moreover, the simple protocol developed here for measuring the absolute temperature inside a single cell using a single FND enables successful temperature measurement in a cell with an accuracy better than ±1°C.

Tech Support

Original Source

DOI: https://doi.org/10.2142/biophysico.15.0_229