The Basics of Microinjection
The microinjection technique is a valuable tool for many applications in cell biology and other disciplines. This technique is used to inject small molecules, such as dyes and mRNAs into single cells, embryos, and other complex tissues. Microinjection has become a critical tool for the study of many biological processes, including cell differentiation and developmental processes such as synapse formation. It also provides a tool for genetic manipulation of embryos and other organisms. This method of cell microinjection is very versatile and can be used with a wide variety of organisms, from Xenopus eggs to human and animal embryos.
Microinjection is a technique in which a liquid is injected into a living tissue using a micropipette. The precise volume of the injection is controlled by pressure pulses of air, which can be adjusted by the user. The microinjection system is equipped with a pipette holder that secures the micropipette for use during the procedure and an airline that provides the compressed air. The microinjection system can be operated by novice researchers, since it requires no special training and is easy to set up and operate.
One of the most important steps in performing microinjection is locating the target region of the tissue under the microscope. The user annotates a location in the field of view (FOV) that contains the desired target, such as the nucleus of a cell. This annotation is recorded by the computer, and the corresponding coordinates are passed to the microinjection system, which uses these coordinates to align the pipette with the target area. Once the coordinates are aligned, the microinjection system generates the necessary pressure pulses and applies them to the pipette.
The pulsative flow patterns of the passive microinjection system are carefully synchronized to steer Droplet during the injection process and to fill it with Injected during the resting step. The amplitudes of the pulsative flows are chosen so that they do not interfere with each other during the injection cycle, but still produce a well-steered double emulsion. When the injection has finished, the pulsative flow patterns are reversed to provide backpressure that pulls the double emulsion off the microneedle and into the downstream microchannel.
In order to guarantee accurate positioning of the micropipette, the system is calibrated before each experiment. This is accomplished by determining the position of the tip of the micropipette within the FOV of the microscope. The calibration procedure is performed by three experimenters with no prior experience in the use of the Autoinjector. Each experimenter annotates the position of the micropipette at different points in the FOV during the calibration process. The results from these annotated points are then compared with the predicted position of the micropipette, and the correct positions of the micropipette for each point in the FOV are determined.
The next step in the microinjection process is to prepare the embryo for injection. The embryo is then lowered into the field of view of the microscope and rotated so that the blastomere, a grainy, yellow bump of cytoplasm located on the top of the yolk, is directly perpendicular to the end of the microinjection needle. Then, the microinjection needle is inserted through the blastomere and the solution is injected into the embryo. After the microinjection, most embryos will survive and develop to the two-cell stage, but some will not. These embryos will not divide correctly and will exhibit a fragmented appearance, indicating that they have been irreversibly damaged by the injection. micro injection