rus
Issue #3-4/2025
O.V.Ivanov, A.I.Akhmetova, I.V.Yaminsky
DEVELOPMENT OF A BIOCOMPATIBLE MULTI-ELECTRODE CELL FOR STUDYING LIVING NEURONAL NETWORKS USING COMBINED SCANNING CAPILLARY MICROSCOPY
DEVELOPMENT OF A BIOCOMPATIBLE MULTI-ELECTRODE CELL FOR STUDYING LIVING NEURONAL NETWORKS USING COMBINED SCANNING CAPILLARY MICROSCOPY
DOI: https://doi.org/10.22184/1993-8578.2025.18.3-4.166.172
An innovative biocompatible multielectrode cell integrating neuronal electrical activity recording technologies with high-resolution scanning capillary microscopy capabilities has been developed and characterized. The proposed design includes an ITO-coated glass substrate with laser-patterned electrodes insulated with a 180 nm thick parylene layer and functionalized copper-gold microelectrodes with memristor properties. The prototype demonstrates the advantages of simultaneous recording of neuronal electrical activity and morphological changes, opening up new possibilities for studying neuronal plasticity, structural distribution of nervous tissue, and screening of neuroactive compounds.
An innovative biocompatible multielectrode cell integrating neuronal electrical activity recording technologies with high-resolution scanning capillary microscopy capabilities has been developed and characterized. The proposed design includes an ITO-coated glass substrate with laser-patterned electrodes insulated with a 180 nm thick parylene layer and functionalized copper-gold microelectrodes with memristor properties. The prototype demonstrates the advantages of simultaneous recording of neuronal electrical activity and morphological changes, opening up new possibilities for studying neuronal plasticity, structural distribution of nervous tissue, and screening of neuroactive compounds.
Теги: biocompatible multielectrode cell electrophysiology neurons scanning capillary microscopy биосовместимая мультиэлектродная ячейка нейроны сканирующая капиллярная микроскопия электрофизиология
INTRODUCTION
Cells for 3D imaging and electrophysiology. Review
Modern neurobiological research requires integration of electrophysiological and microscopic methods for a comprehensive analysis of the structural and functional neural networks properties. Existing solutions in this field have various advantages and limitations.
Commercial Multielectrode systems, like Multielectrode Arrays or MEA systems, with transparent substrates, such as the MEA2100-System from Multichannel Systems, allow electrophysiological recording to be combined with inverted optical microscopy [1]. However, these systems are hardly compatible with high-resolution microscopy techniques due to the large substrate thickness and the complex electrical signal recording system, as well as a significant working distance.
Experimental developments include systems with integrated fibre optic sensors that allow optical stimulation and recording directly at the area where the MEA electrodes are located. Such systems provide spatially coupled electrical and optical recording, but have limited spatial resolution and require complex manufacturing processes.
To overcome optical limitations, MEAs based on transparent conducting materials have been proposed. For example, Park et al. developed MEAs with graphene electrodes providing optical transparency >90% in the visible range [2]. However, the used graphene electrodes have a relatively high impedance (>1 megohm at 1 kHz), which limits their application for recording low-amplitude signals.
A promising direction is integration of MEA with scanning probe microscopy methods. Systems combining MEA registration with atomic force microscopy make it possible to study correlation between morphological and electrical changes in neurons. However, these systems have limitations related to the geometry and electrical isolation of AFM probes. There is also some difficulty in combining the measuring system (usually optical) of AFM with the liquid medium of cultivation. In this context, the developed biocompatible cell offers a fundamentally new approach combining the capillary microscopy advantages and high-density MEA.
Development of a cell for 3D visualisation of bio-objects with simultaneous electrophysiological measurements
Substrate and electrode system
The developed biocompatible multi-electrode cell is based on a 50 × 50 × 1 mm3 glass substrate coated with indium tin oxide (ITO, Indium Tin Oxide). ITO was chosen due to its optical transparency (>85% in the visible range) combined with good electrical conductivity (surface resistivity <10 Ohm/square), which ensures compatibility with optical microscopy methods [3–5].
The electrode system structure is formed by laser annealing using SharpLase SHARPMARK 20-F with Nd:YAG laser (λ=1064 nm, power 20 W) in pulse mode with "High contrast" option. This method provides spatial resolution of positioning ~10 µm, which is sufficient for production of precision microelectrode structures. The advantage of laser annealing of macroelectrodes over traditional photolithography is significant efficiency of the technique and high speed of substrate fabrication without the use of chemical reagents.
The electrode system includes four macroelectrodes (5 × 10 mm) at the periphery of the substrate to interface with the recording equipment, current carrying tracks with a 1 mm gap between them and maximum coverage area to increase the total cell capacitance, reduce the resistance of each electrode, and four microelectrodes (300 µm) in the centre of the top layer of the cell.
The electrode configuration is optimised to provide maximum spatial resolution while minimising electromagnetic interference between recording channels.
Insulation and operating of electrodes
The 180 nm thick parylene-C layer is used to electrically isolate the current carrying tracks and define the active zones of the microelectrodes (Fig.1). The parylene layer was deposited by surface polymerisation in the gas phase using an SCS Labcoater PDS 2010 vacuum sputtering system. The PDS 2010 converts a parylene dimer (2,2-paracyclophane or its derivatives) into a gaseous monomer. The material polymerises on the substrate when it is applied at room temperature. At the vacuum levels used, the conductive layer of the substrate was uniformly treated with the gaseous monomer followed by deposition, resulting in a durable protective coating.
The working microelectrodes are formed by magnetron sputtering of copper and gold sequentially on the parylene surface through a mask with 300 × 300 μm holes. The first sputtering layer is made by copper with a thickness of about 300 nm for subsequent filament formation in the parylene layer. The above metals were chosen because of their wide application in electronic engineering, including the fabrication of memristive devices [6]. Gold was chosen as the electrode material due to its high biocompatibility, corrosion resistance in physiological solutions and low impedance at the electrode-electrolyte interface. Copper was chosen as an electrochemically active metal to form the filaments through the layer of parylene insulator by analogy with the technology of filament formation in the manufacturing of neuromorphic systems [7].
An innovative aspect of the developed cell is application of conductive filament formation technology to make an electrical contact between platinum microelectrodes and underlying ITO electrodes through a parylene layer. This process is carried out by applying a controlled voltage (5–7 V) between the platinum microelectrode and the corresponding ITO contact with current limitation at 100 μA. This leads to a local electrical breakdown of parylene and preparing of a nanometre conductive channel with a controlled ohmic characteristic [8].
The peculiarity of the formed filaments is possibility of controlled change of their conductivity depending on the applied voltage, which can be used to simulate synaptic plasticity in development of biomimetic neurointerfaces. In addition, the nonlinear conductivity of filaments provides natural filtration of high-frequency components of recorded signals, improving the signal-to-noise ratio when recording action potentials. At the same time, parylene has important properties of biocompatibility, is chemically inert, and has a flexible set of functional properties [9].
Integration of the cultural chamber
A modified 40 mm diameter polystyrene Petri dish is used to make the cultural chamber (Fig.2). Modification is performed by mechanical removal of the bottom of the cup by abrasive blasting and subsequent finishing with a surgical scalpel to ensure an even edge. The obtained polymer ring is fixed on the surface of the electrode substrate using UV-curable acrylic resin, which, due to its biocompatibility, is successfully used in development of commonly available commercial cell variants [10]. The curing process is carried out using UV radiation (λ=365 nm, power 100 mW/cm2) for 10 minutes, which ensures complete polymerisation of the adhesive and minimises the amount of unreacted monomers.
The height of the culture chamber is 5 mm, providing a total available volume of about 6 mL with a cultivation area of ~12.6 cm2. This configuration is optimised to maintain a stable volume of culture medium while minimising the gap between the medium surface and the microscope objective, which may be important for high-resolution optical microscopy techniques for initial probe alignment.
Integration of the neuronal viability maintenance system
For long-term in vitro studies of living neural networks, it is important to reproduce physiological conditions, including the temperature regime (37 °C) and gas composition of the medium (5% CO2) [11].
The system of gas environment maintenance is based on continuous supply of prepared gas mixture through the cell with culture. The gas flow is regulated by a rotameter connected to a cylinder with a reducer, which allows precise dosage of gas composition and speed of its supply to the cell. Continuous gas supply provides maintenance of constant CO2 concentration in the contact zone between liquid and gas medium and allows to compensate for leaks occurring through the technological holes of the capillary microscope.
The microfluidic system includes a MasterFlex C/L peristaltic pump, a thermostated buffer tank, and a closed circulation loop of FedroTek biocompatible tubing. The solution is heated to 37 °C by an isolated element with PID control, which eliminates temperature gradients and allows for a system with high precision temperature stabilisation. Peristalsis provides pulsatile flow at a rate of 50–200 µl/min, mimicking natural tissue perfusion, with the cell volume and inlet/outlet valve arrangement within the cell volume to make laminar flow, which is critical for maintaining cell culture viability. Integration of both subsystems into a microscopic complex allows long-term monitoring of morphofunctional dynamics of neurons without disturbing physicochemical parameters of the medium.
CONCLUSIONS
The developed biocompatible multi-electrode cell opens new possibilities to study the neural network dynamics in vitro. Key advantages of the system:
Multimodal recording: Synchronous recording of electrical activity (10 kHz sampling rate) and SCM imaging with electrical bandwidth in the range 0–10 kHz, (1 frame in 5 minutes at a field of 50 × 50 μm2) allow real-time correlation of functional and structural changes.
Biomimetic interface: Memristor filaments mimic the non-linear properties of synapses, enabling two-way interaction with the neural network.
High reproducibility: all components to build the cell are commercially available.
Promising applications include:
Development of biochips for Alzheimer’s disease drug screening with ability to track β-amyloid-induced synaptic dysfunction;
study of the mechanisms of epileptogenesis through spatial and temporal analysis of the spread of pathological activity;
development of new generation neurointerfaces with feedback based on morphofunctional parameters.
ACKNOWLEDGEMENTS
This work was performed under the state order of the Lomonosov Moscow State University. FemtoScan Online software is provided by "Advanced Technologies Center", www.femtoscan.ru
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Cells for 3D imaging and electrophysiology. Review
Modern neurobiological research requires integration of electrophysiological and microscopic methods for a comprehensive analysis of the structural and functional neural networks properties. Existing solutions in this field have various advantages and limitations.
Commercial Multielectrode systems, like Multielectrode Arrays or MEA systems, with transparent substrates, such as the MEA2100-System from Multichannel Systems, allow electrophysiological recording to be combined with inverted optical microscopy [1]. However, these systems are hardly compatible with high-resolution microscopy techniques due to the large substrate thickness and the complex electrical signal recording system, as well as a significant working distance.
Experimental developments include systems with integrated fibre optic sensors that allow optical stimulation and recording directly at the area where the MEA electrodes are located. Such systems provide spatially coupled electrical and optical recording, but have limited spatial resolution and require complex manufacturing processes.
To overcome optical limitations, MEAs based on transparent conducting materials have been proposed. For example, Park et al. developed MEAs with graphene electrodes providing optical transparency >90% in the visible range [2]. However, the used graphene electrodes have a relatively high impedance (>1 megohm at 1 kHz), which limits their application for recording low-amplitude signals.
A promising direction is integration of MEA with scanning probe microscopy methods. Systems combining MEA registration with atomic force microscopy make it possible to study correlation between morphological and electrical changes in neurons. However, these systems have limitations related to the geometry and electrical isolation of AFM probes. There is also some difficulty in combining the measuring system (usually optical) of AFM with the liquid medium of cultivation. In this context, the developed biocompatible cell offers a fundamentally new approach combining the capillary microscopy advantages and high-density MEA.
Development of a cell for 3D visualisation of bio-objects with simultaneous electrophysiological measurements
Substrate and electrode system
The developed biocompatible multi-electrode cell is based on a 50 × 50 × 1 mm3 glass substrate coated with indium tin oxide (ITO, Indium Tin Oxide). ITO was chosen due to its optical transparency (>85% in the visible range) combined with good electrical conductivity (surface resistivity <10 Ohm/square), which ensures compatibility with optical microscopy methods [3–5].
The electrode system structure is formed by laser annealing using SharpLase SHARPMARK 20-F with Nd:YAG laser (λ=1064 nm, power 20 W) in pulse mode with "High contrast" option. This method provides spatial resolution of positioning ~10 µm, which is sufficient for production of precision microelectrode structures. The advantage of laser annealing of macroelectrodes over traditional photolithography is significant efficiency of the technique and high speed of substrate fabrication without the use of chemical reagents.
The electrode system includes four macroelectrodes (5 × 10 mm) at the periphery of the substrate to interface with the recording equipment, current carrying tracks with a 1 mm gap between them and maximum coverage area to increase the total cell capacitance, reduce the resistance of each electrode, and four microelectrodes (300 µm) in the centre of the top layer of the cell.
The electrode configuration is optimised to provide maximum spatial resolution while minimising electromagnetic interference between recording channels.
Insulation and operating of electrodes
The 180 nm thick parylene-C layer is used to electrically isolate the current carrying tracks and define the active zones of the microelectrodes (Fig.1). The parylene layer was deposited by surface polymerisation in the gas phase using an SCS Labcoater PDS 2010 vacuum sputtering system. The PDS 2010 converts a parylene dimer (2,2-paracyclophane or its derivatives) into a gaseous monomer. The material polymerises on the substrate when it is applied at room temperature. At the vacuum levels used, the conductive layer of the substrate was uniformly treated with the gaseous monomer followed by deposition, resulting in a durable protective coating.
The working microelectrodes are formed by magnetron sputtering of copper and gold sequentially on the parylene surface through a mask with 300 × 300 μm holes. The first sputtering layer is made by copper with a thickness of about 300 nm for subsequent filament formation in the parylene layer. The above metals were chosen because of their wide application in electronic engineering, including the fabrication of memristive devices [6]. Gold was chosen as the electrode material due to its high biocompatibility, corrosion resistance in physiological solutions and low impedance at the electrode-electrolyte interface. Copper was chosen as an electrochemically active metal to form the filaments through the layer of parylene insulator by analogy with the technology of filament formation in the manufacturing of neuromorphic systems [7].
An innovative aspect of the developed cell is application of conductive filament formation technology to make an electrical contact between platinum microelectrodes and underlying ITO electrodes through a parylene layer. This process is carried out by applying a controlled voltage (5–7 V) between the platinum microelectrode and the corresponding ITO contact with current limitation at 100 μA. This leads to a local electrical breakdown of parylene and preparing of a nanometre conductive channel with a controlled ohmic characteristic [8].
The peculiarity of the formed filaments is possibility of controlled change of their conductivity depending on the applied voltage, which can be used to simulate synaptic plasticity in development of biomimetic neurointerfaces. In addition, the nonlinear conductivity of filaments provides natural filtration of high-frequency components of recorded signals, improving the signal-to-noise ratio when recording action potentials. At the same time, parylene has important properties of biocompatibility, is chemically inert, and has a flexible set of functional properties [9].
Integration of the cultural chamber
A modified 40 mm diameter polystyrene Petri dish is used to make the cultural chamber (Fig.2). Modification is performed by mechanical removal of the bottom of the cup by abrasive blasting and subsequent finishing with a surgical scalpel to ensure an even edge. The obtained polymer ring is fixed on the surface of the electrode substrate using UV-curable acrylic resin, which, due to its biocompatibility, is successfully used in development of commonly available commercial cell variants [10]. The curing process is carried out using UV radiation (λ=365 nm, power 100 mW/cm2) for 10 minutes, which ensures complete polymerisation of the adhesive and minimises the amount of unreacted monomers.
The height of the culture chamber is 5 mm, providing a total available volume of about 6 mL with a cultivation area of ~12.6 cm2. This configuration is optimised to maintain a stable volume of culture medium while minimising the gap between the medium surface and the microscope objective, which may be important for high-resolution optical microscopy techniques for initial probe alignment.
Integration of the neuronal viability maintenance system
For long-term in vitro studies of living neural networks, it is important to reproduce physiological conditions, including the temperature regime (37 °C) and gas composition of the medium (5% CO2) [11].
The system of gas environment maintenance is based on continuous supply of prepared gas mixture through the cell with culture. The gas flow is regulated by a rotameter connected to a cylinder with a reducer, which allows precise dosage of gas composition and speed of its supply to the cell. Continuous gas supply provides maintenance of constant CO2 concentration in the contact zone between liquid and gas medium and allows to compensate for leaks occurring through the technological holes of the capillary microscope.
The microfluidic system includes a MasterFlex C/L peristaltic pump, a thermostated buffer tank, and a closed circulation loop of FedroTek biocompatible tubing. The solution is heated to 37 °C by an isolated element with PID control, which eliminates temperature gradients and allows for a system with high precision temperature stabilisation. Peristalsis provides pulsatile flow at a rate of 50–200 µl/min, mimicking natural tissue perfusion, with the cell volume and inlet/outlet valve arrangement within the cell volume to make laminar flow, which is critical for maintaining cell culture viability. Integration of both subsystems into a microscopic complex allows long-term monitoring of morphofunctional dynamics of neurons without disturbing physicochemical parameters of the medium.
CONCLUSIONS
The developed biocompatible multi-electrode cell opens new possibilities to study the neural network dynamics in vitro. Key advantages of the system:
Multimodal recording: Synchronous recording of electrical activity (10 kHz sampling rate) and SCM imaging with electrical bandwidth in the range 0–10 kHz, (1 frame in 5 minutes at a field of 50 × 50 μm2) allow real-time correlation of functional and structural changes.
Biomimetic interface: Memristor filaments mimic the non-linear properties of synapses, enabling two-way interaction with the neural network.
High reproducibility: all components to build the cell are commercially available.
Promising applications include:
Development of biochips for Alzheimer’s disease drug screening with ability to track β-amyloid-induced synaptic dysfunction;
study of the mechanisms of epileptogenesis through spatial and temporal analysis of the spread of pathological activity;
development of new generation neurointerfaces with feedback based on morphofunctional parameters.
ACKNOWLEDGEMENTS
This work was performed under the state order of the Lomonosov Moscow State University. FemtoScan Online software is provided by "Advanced Technologies Center", www.femtoscan.ru
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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