Nanodiamond–Quantum Sensors Reveal Temperature Variation Associated to Hippocampal Neurons Firing

At a Glance

Metadata	Details
Publication Date	2022-07-25
Journal	Advanced Science
Authors	Giulia Petrini, Giulia Tomagra, Ettore Bernardi, Ekaterina Moreva, P. Traina
Institutions	Torino e-district, Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry
Citations	52
Analysis	Full AI Review Included

Executive Summary

This research utilizes nanodiamond (ND) quantum sensors to perform highly localized intracellular thermometry, establishing a direct correlation between neuronal firing activity and temperature fluctuations in hippocampal neurons.

Core Achievement: First-time detection and quantification of temperature variations (up to 1 °C) associated with the potentiation and depletion of neuronal firing activity.
Sensing Technology: Nitrogen-Vacancy (NV) centers in internalized nanodiamonds, interrogated via Optically Detected Magnetic Resonance (ODMR).
Key Findings: Neuronal firing potentiation (using Picrotoxin) caused a significant temperature increase of 1.02 ± 0.24 °C. Silencing the network (using TTX+Cd) resulted in a temperature decrease of 0.50 ± 0.17 °C.
Performance Metrics: Demonstrated measurement sensitivity capable of discriminating variations of 0.5 °C, with a projected sensitivity of less than 0.1 °C.
Biocompatibility: The ND internalization protocol (0.6 µg/ml concentration) was confirmed to be non-neurotoxic, preserving cell excitability and action potential waveforms.
Implication: Provides a robust, nanoscale tool for assessing neuronal spiking activity and studying localized temperature gradients generated by metabolic processes under physiological and pathological conditions.

Technical Specifications

Parameter	Value	Unit	Context
ND Sensor Type	NV^- Centers	-	Nanodiamond quantum thermometer
ND Hydrodynamic Diameter	185	nm	Average size used for cellular internalization
ND Incubation Concentration	0.6	µg/ml	Used for 5-hour incubation
Cytotoxicity Threshold (ND)	> 250	µg/ml	Concentration far below toxic levels
Bath Temperature (T_bath)	37.0 ± 0.1	°C	Controlled incubation chamber temperature
Temperature Increase (+PICRO)	1.02 ± 0.24	°C	Associated with potentiated firing
Temperature Decrease (+TTX+Cd)	-0.50 ± 0.17	°C	Associated with silenced firing
ODMR Coupling Constant (dDgs/dT)	-76 ± 4	kHz/°C	Calibrated value for NDs (weighted mean)
Laser Excitation Wavelength	532	nm	CW laser (Coherent Prometheus 100NE)
Laser Power (Attenuated)	1	mW	Used to excite NV centers
Microwave Power (Amplified)	20	dBm	Fed to planar broadband antenna
Objective Specification	60x, NA = 0.67	-	Olympus UPLANFL air objective
Measurement Acquisition Time	60	s	Used for PL measurements (trade-off for precision)
Demonstrated Sensitivity	0.5	°C	Minimum variation discriminated by the sensor
Projected Sensitivity	< 0.1	°C	Potential sensitivity of the technique
ND Sensor Sensitivity (η)	~3	°C/sqrt(Hz)	Estimated sensitivity based on calibration

Key Methodologies

The experiment relies on a highly controlled ODMR setup integrated into a confocal microscope, combined with specific chemical modulation of neuronal activity.

Nanodiamond (ND) Preparation:
- Monodisperse NDs (MSY 0-0.25) were oxidized (510 °C in air, followed by wet oxidation in HF:HNO₃).
- NV centers were created by irradiation (15.7 MeV electron beam at 870 °C) and subsequent annealing (900 °C under argon).
- Final hydrodynamic diameter was 205 nm.
Cell Culture and Labeling:
- Hippocampal neurons (10 Days In Vitro, DIV) were plated on Petri dishes.
- Cells were incubated for 5 hours with 0.6 µg/ml NDs to facilitate internalization.
- Confocal imaging confirmed ND internalization and verified that the protocol did not affect cell excitability or ion channel function.
ODMR Experimental Setup:
- The setup used an Olympus IX73 inverted microscope with integrated single-photon confocal imaging and microwave control.
- A 532 nm laser (attenuated to 1 mW) excited the NV centers. Laser emission was controlled by an Acousto-Optic Modulator (AOM).
- Microwave radiation (20 dBm) was delivered via a homemade planar broadband antenna placed beneath the Petri dish.
- The sample was maintained at 37.0 ± 0.1 °C in a closed incubation chamber.
Thermometry Protocol (Differential ODMR):
- The ODMR spectrum was acquired, and the microwave frequency was set to the resonant frequency (Dgs).
- Temperature variation (ΔT) was determined by measuring the shift in the photoluminescence (PL) signal (ΔF) at the resonant frequency.
- The relationship ΔF = slope · (dDgs/dT) · ΔT was used, where the coupling constant dDgs/dT was independently calibrated as -76 kHz/°C.
Neuronal Activity Modulation Conditions:
- Control (CTRL): Measurements taken before and after perfusion with saline Tyrode solution (spontaneous firing).
- Potentiation (+PICRO): Perfusion with 100 µM Picrotoxin (GABA_A inhibitor) to drastically increase firing rate.
- Silencing (+TTX+Cd): Subsequent perfusion with 0.3 µM Tetrodotoxin (TTX) and 500 µM Cadmium Chloride (Cd) to block Na⁺ and Ca²⁺ channels, depleting firing activity.

Commercial Applications

The development of biocompatible, nanoscale quantum sensors for intracellular thermometry has significant implications across several high-tech and biomedical sectors.

Quantum Sensing and Metrology:
- Advancement of NV-center technology for high-sensitivity, non-invasive measurements in complex biological environments.
- Development of integrated quantum-classical systems (e.g., combining ODMR with MEAs) for synchronized electrical and metabolic monitoring.
Neuroscience and Physiology:
- Real-time mapping of localized thermogenesis associated with action potential generation, synaptic transmission, and maintenance of membrane potentials.
- Systematic study of energy consumption and metabolic efficiency in neuronal networks at the subcellular level.
Drug Discovery and Pharmacology:
- Tool for assessing the metabolic impact of pharmaceutical compounds (e.g., Picrotoxin) on cellular activity, providing data beyond traditional electrical or chemical assays.
Cellular Diagnostics and Pathology:
- Monitoring localized temperature gradients as biomarkers for pathological conditions characterized by altered metabolism, such as cancer (high metabolic activity) or neurodegenerative diseases (e.g., Alzheimer’s, Parkinson’s).
Biomaterials and Nanotechnology:
- Engineering functionalized nanodiamonds for targeted delivery to specific subcellular compartments (e.g., mitochondria) to detect thermogenesis at specific organelles.

View Original Abstract

Abstract Temperature is one of the most relevant parameters for the regulation of intracellular processes. Measuring localized subcellular temperature gradients is fundamental for a deeper understanding of cell function, such as the genesis of action potentials, and cell metabolism. Notwithstanding several proposed techniques, at the moment detection of temperature fluctuations at the subcellular level still represents an ongoing challenge. Here, for the first time, temperature variations (1 °C) associated with potentiation and inhibition of neuronal firing is detected, by exploiting a nanoscale thermometer based on optically detected magnetic resonance in nanodiamonds. The results demonstrate that nitrogen‐vacancy centers in nanodiamonds provide a tool for assessing various levels of neuronal spiking activity, since they are suitable for monitoring different temperature variations, respectively, associated with the spontaneous firing of hippocampal neurons, the disinhibition of GABAergic transmission and the silencing of the network. Conjugated with the high sensitivity of this technique (in perspective sensitive to < 0.1 °C variations), nanodiamonds pave the way to a systematic study of the generation of localized temperature gradients under physiological and pathological conditions. Furthermore, they prompt further studies explaining in detail the physiological mechanism originating this effect.

Tech Support

Original Source

DOI: https://doi.org/10.1002/advs.202202014

References

2002 - Quantification of Cyclin B1 and P34cdc2 in Bovine Cumulus‐Oocyte Complexes and Expression Mapping of Genes Involved in the Cell Cycle by Complementary DNA Macroarrays