Why is membrane focusing critical before microinjection?

Focusing on the membrane separating follicle and nurse cells ensures accurate needle placement and prevents damage to surrounding structures during injection.

How does developmental stage selection affect microinjection success?

Mid to late-stage egg chambers provide large, visible nurse cells and oocyte yolk, improving visibility and injection accuracy for consistent probe delivery.

What role does compensation pressure play in the microinjection process?

Compensation pressure maintains continuous solution flow from the needle, preventing clogging and ensuring reproducible injection volumes during molecular beacon delivery.

Why is needle cleaning performed before injection?

Performing a clean function removes air from the needle tip, which could block flow or cause inconsistent delivery of the molecular beacon solution.

How does post-injection objective switching support imaging workflows?

Changing to the desired magnification after injection enables optimized image acquisition of transfected nurse cells without disturbing the sample.

Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery

JoVE Encyclopedia of Experiments

Biology

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- Place a coverslip with dissected egg chambers onto the microscope stage. Locate an egg chamber of the appropriate developmental stage and orientation for microinjection. Mid to late-stage egg chambers have a large visible oocyte yolk, and those in the correct orientation are with their anterior-posterior axis perpendicular to the direction of injection.

Drosophila nerve cells are a group of 15 cells inside the egg chamber responsible for depositing maternally derived nutrients and biomolecules into the developing oocyte. Set up the microinjector system to the appropriate injection and compensation pressure to ensure consistent injection volume and outward flow. Carefully bring a previously loaded capillary needle into the field of view so that the cells and needle are in the same focal plane.

Ensure that there is no air trapped in the tip of the capillary and that there is a continuous flow of the solution. Then adjust the Z-position of the objective to focus on the membrane separating the follicle cells from the nurse cells, insert the needle into a nurse cell and perform the injection.

The following protocol demonstrates microinjecting Drosophila nurse cells with fluorescent oligonucleotide probes, called molecular beacons, against maternally contributed mRNA transcripts for live cell imaging.

- For molecular beacon microinjection, select the 40x oil objective and mount a cover slip with the dissected egg chamber onto the microscope stage. Locate an egg chamber at the mid to late developmental stage that is properly oriented for microinjection, then set up a microinjector with the appropriate injection and compensation pressures.

Using the micromanipulator joystick, gently lower a needle loaded with one microliter of molecular beacon solution into the oil drop. Bring the tip into focus toward the periphery of the field of view. Perform a clean function to remove the air from the needle tip and to ensure that there is flow from the needle, and bring the needle to the home position.

Focus on the egg chamber to be microinjected, and bring the needle back into focus near the edge of the egg chamber. Perform a fine adjustment of the Z-position of the objective such that the membrane separating the follicle cells from the nurse cells is in focus. Insert the needle into a nurse cell for injection of the molecular beacons over a period of two to five seconds.

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Last updated: 4 July 2026

Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery

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