All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

Neuronal firing patterns are the result of inputs converging onto single cells. Identifying these inputs, anatomically and functionally, is essential to understand how neurons integrate information. Single-cell electroporation of helper genes and subsequent local injection of recombinant rabies viruses enable precise mapping of inputs to individual cells in superficial layers of the intact cortex. However, access to neurons in deeper structures requires more invasive procedures, including removal of overlying tissue. We have developed a method that through a combination of virus injections allows us to target ≤4 hippocampal cells 48% of the time and a single cell 16% of the time in wildtype mice without the use of electroporation or tissue aspiration. We identify local and distant monosynaptic inputs that can be functionally characterised in vivo. By expanding the toolbox for monosynaptic circuit tracing, this method will help further our understanding of neuronal integration at the level of single cells.

Deep Learning and Computer Vision Strategies for Automated Gene Editing with a Single-Cell Electroporation Platform

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9–guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein–encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.

Simulation of single cell electroporation

This paper attempts to introduce the dynamics of electroporation into the single cell model. The main characteristics of the model were described. Results show the generation and rise of pores in a round cell with a radius of 50 μm exposed to 40 kV/m electric field for 1 ms were analysed, so that the transmembrane potential, the number of pores and the membrane conductance could be calculated. Finally, how the model can help explain the experiment is discussed.

Single-Cell Electroporation with Real-Time Impedance Assessment Using a Constriction Microchannel

The electroporation system can serve as a tool for the intracellular delivery of foreign cargos. However, this technique is presently limited by the inaccurate electric field applied to the single cells and lack of a real-time electroporation metrics subsystem. Here, we reported a microfluidic system for precise and rapid single-cell electroporation and simultaneous impedance monitoring in a constriction microchannel. When single cells (A549) were continuously passing through the constriction microchannel, a localized high electric field was applied on the cell membrane, which resulted in highly efficient (up to 96.6%) electroporation. During a single cell entering the constriction channel, an abrupt impedance drop was noticed and demonstrated to be correlated with the occurrence of electroporation. Besides, while the cell was moving in the constriction channel, the stabilized impedance showed the capability to quantify the electroporation extent. The correspondence of the impedance variation and electroporation was validated by the intracellular delivery of the fluorescence indicator (propidium iodide). Based on the obtained results, this system is capable of precise control of electroporation and real-time, label-free impedance assessment, providing a potential tool for intracellular delivery and other biomedical applications.