Intracellular delivery and voltage sensitivity of nanomaterials for the optical imaging of neuronal activity

Abstract

Monitoring the electrical signals generated by neurons to transmit information, is central to understanding how the brain and nervous systems work. The photoluminescence (PL) of some nanomaterials, such as semiconductor quantum dots (QDs) and fluorescent nanodiamonds (NDs), has shown higher sensitivity to electrical fields than that of any previously reported probes, which may address the persistent challenge of robust optical voltage imaging. The fundamental issue for implementing voltage-sensitive nanomaterials (VSM) in live neurons is their delivery into the plasma membrane bilayer. Currently, the delivery has been demonstrated on QDs via their spontaneous insertion directly into the plasma membrane bilayer, or indirectly into the bilayer of liposomes that later fuse with the plasma membrane. In both methods, QDs are introduced from the extracellular space and implemented to image the activity of neuronal assemblies. The first part of this thesis explores the implementation of VSM in another scenario, i.e., the voltage imaging from multiple sites on single neurons. After direct intracellular delivery, amphiphilic nanomaterials are expected to spread into distal processes and insert into the plasma membrane bilayer, being able to monitor the electrical activity in the smallest neuronal structures, such as dendritic spines. Here, the intracellular delivery of nontargeted QDs as an example, has been demonstrated by microelectrophoresis technique, where electrical currents were applied to eject charged QDs through fine-tipped glass micropipettes into living cells. The amount of delivered QDs was finely controlled by tuning the ejection duration, which had a substantial impact on preserving short-term and long-term cell health. Delivered QDs were homogeneously distributed throughout the cytoplasm and presented pure Brownian diffusion without endosomal entrapment. These original and promising results lay the foundation to apply the microelectrophoresis technique to other VSM, including the protocol for preparing nanomaterials suspension and the required tip sizes of micropipettes, which are key to their successful intracellular loading. Another fundamental issue is ascertaining the PL responses of these nanomaterials to applied voltage modulations. The second part of this thesis describes the fabrication of a multilayer device that can apply a homogeneous electric field to the embedded nanomaterials (NDs as an example). By using ultrasonication, NDs were well dispersed as single particles within the device, where the PL responses of individual NDs can be examined. Other fabrication details, such as film thickness and electrode deposition, were also described. These results provide a high-throughput screening platform to characterize the voltage sensitivities of different nanomaterials, which helps to iteratively improve their design and synthesis, including composition, size, shape, and band alignment. Collectively, the findings in this thesis provide a significant contribution to the unique interface of neuroscience and nanomaterials regarding the optical visualization of neuronal activity. The pioneering work here facilitates the future use of microelectrophoresis technique to deliver various VSM for multisite voltage imaging of single neurons. The deployment of the multilayer device promotes the development and optimization of new nanomaterials with enhanced voltage sensitivity. With these fundamental challenges to be addressed in the near future, real-time in vivo voltage imaging may be attainable in relevant animal models to elucidate the complex function of brain and nervous systems.