Image-Guided Robotic Cell Microinjection: An Automated High-Throughput Technique for the Precise Delivery of Fluorescently-Labeled Polysaccharides Into Single Neuronal Cells in Organotypic Brain Slice Cultures
Transcrição
To perform robotic cell microinjection, begin by loading a chilled, fluorescently-labeled biocompatible polysaccharide solution into an optimized microinjection needle. Mount the loaded needle onto the holder of a programmable micromanipulator for precise needle positioning during microinjection.
Transfer a culture dish containing mouse embryonic organotypic brain tissue slices in media on a heated microscope stage to maintain tissue physiological conditions. Focus on the basal surface of the brain tissue slice, which comprises pyramidal neurons, and immerse the needle in media to position it close to the neurons to be microinjected.
Adjust compensation pressure to prevent media backflow into the needle via capillary action.
Using software, define the trajectory and needle angle. Position the needle at the start-point of the mapped trajectory and initiate the automated microinjection run.
The needle travels a specified distance towards the target cell. The applied force causes the needle to press against the cell membrane, which builds up local tension, and rupture the phospholipid bilayer, allowing penetration. Upon access to the cytoplasmic compartment, the needle – as per set injection pressure – dispenses a controlled volume of the loaded solution. Post-injection, the needle retracts.
The perforation in the cell membrane elicits an intrinsic cellular repair response to restore membrane homeostasis. The cell membrane reseals with minimal damage to the neurons, entrapping the fluorescently-labeled polysaccharide solution. The microinjection run continues until all cells within the trajectory are injected. Successfully injected neurons in brain tissue slices exhibit fluorescence.
