Abstract
Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells’ health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC’s) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.
Highlights
Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells
The amplifier of the Mobile Nanoinjection (MoNa) system turns out to provide a noise level which is roughly tenfold higher compared to the amplifier of our original scanning ion conductance microscopy (SICM) setup, we were able to reliably approach single cells and perform injection of fluorescent probes
We could realize cost reductions of about 85% compared to a full-blown SICM instrument
Summary
Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. The almost unlimited reservoir of molecules from microinjection and the controllable electrokinetic-driven delivery in combination with a precise axial position feedback system provides a method for the fast and easy positioning of the pipette and insertion of molecules - e.g. fluorescent probes - into single living cells. Exploiting the unique approach technique by tracking the ionic current flowing between two electrodes through the tip of the nanopipette, this allows for the precise axial positioning of the nanopipette with respect to the plasma membranes of single cells This enables the location-specific penetration of the membrane and injection of single cells as well as cellular compartments such as the nucleus with molecules. All of the equipment mentioned above is heavy, difficult to set up and lacks flexibility to connect with different workspaces without major adjustments and days of calibration
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