In microinjection, a small amount of liquid is injected into tissue at the microscopic or borderline macroscopic level with the goal of transfecting cells within tissue. This technique is commonly used in embryonic development, cell biology and tissue engineering to deliver reagents to individual cells or cell populations. Although it offers many advantages, the technique is prone to error and requires a high degree of skill to perform reliably. A robotic system that precisely guides the injection micropipette to cells while under microscope guidance can greatly increase the throughput and yield of the microinjection process.

A typical microinjection experiment involves a user guiding the injection micropipette to the surface of the tissue, inserting the tip into the tissue, performing an injection, and then withdrawing the pipette back within a brief period. The depth of tissue penetration and how long the injection needle stays inserted in the cell are critical factors for achieving a successful transfection.

The traditional manual microinjection procedure is time-consuming and labor intensive, requiring a great deal of training and experience to master. Moreover, the process is prone to errors due to the limited spatial and temporal precision available with a human hand and eye. This leads to variation in the quality of experiments performed, which can lead to inconsistent results and poor-quality data.

To overcome these limitations, we have developed a robotic microinjection system that is capable of precisely guiding the injection micropipette into cells under microscope image guidance. This system, which we have named the Autoinjector, utilizes a simple modification of an existing microinjection station and is able to programmatically control both the position of the injection micropipette and the injection pressure (see Fig. 1).

We have utilized the Autoinjector to track neural progenitor cells in vivo and to systematically study gap-junctional communication between neurons in the developing mouse telencephalon. Our results showed that the Autoinjector is able to accurately target neuronal progenitor cells, which can be identified by their Sox2-positive, Cajal-Retzius-negative morphology, and elicit the formation of downstream progeny including pyramidal neurons, as shown in Figs. 6C and EV2. Furthermore, we found that the Autoinjector does not disrupt normal AP-to-BP transitions in a tissue-based model of neural progenitor cell fate tracing.

3.5-cm pieces of brain were cut by hand with a micro-knife and placed in a 37degC warm slice culture medium [SCM: Neurobasal medium (Thermo Fisher Scientific), 10% rat serum, Penstrep supplement, B27 supplement (17502048; Thermo Fisher Scientific), N2 supplement]. Each tissue piece was then covered with a layer of mineral oil to prevent the penetration of the needle. Pseudo cells were induced by applying negative pressure to the outlet of the cell holder chip immersed in mineral oil, and water droplets containing different concentrations of TRITC-dextran were injected through the designed channels in the cell holder. The injected water droplets were analyzed by measuring their size and fluorescence under a series of injection pressures and times.

To generate transgenic mice, we injected a 7 kb neo gene into the male pronucleus of an E12.5 murine embryo using the Autoinjector. The resulting tTACMV/NZL double-transgenic founder mice were crossed with wild-type mice to select for transgenic offspring. PCR-based genotyping confirmed the presence of the neo gene in the offspring.micro injection