Protocol 1: Washing and injection of super-paramagnetic beads

Step 1: Washing the beads
Take 10μl of the stock bead solution (10mg/ml) in an Eppendorf tube. Bring a permanent magnet close to the tube to clump the beads and remove the remaining solution using a pipette. Take the magnet away and resuspend the beads in 20μl of distilled water. Repeat the process twice before injecting the diluted bead solution bead solution (5mg/ml) into embryo.

Step 2: Microinjection
Injections were performed using a micro-injector and injection needles. The opening of the needle is adjusted such that it is not too small for the beads to come out as well as not too large to damage the embryo. Beads are injected at extremely low pressure (~5psi) and long injection duration (~150ms) in order to avoid dispersion of beads in the yolk. Since the beads sink to the tip of the needle and potentially block the needle the pressure had to be increased for some injections (~25psi) to prevent blocking of the needle.

Step 3: Aggregation of injected beads
By applying a strong constant magnetic field after the injection of the beads with a permanent magnet the beads are attracted and clump. This aggregation of beads preserves the single beads from translating through the yolk and eases the rotation.

Step 4a: Embedding of zebrafish embryo for SPIM experiments
The zebrafish embryo in E3 buffer is sucked up into a FEP tube with a syringe. The inner tube diameter (1.0 mm) is chosen a bit smaller than the zebrafish chorion (1.2 mm) that the fish is not pulled out of the tube by the lower magnet. The embryo (0.8 mm) is still free to move within its chorion.

Step 4b: Embedding of zebrafish larvae (5 dpf) for spinning disk experiments
For coating the inner surface of the FEP tubes with methyl cellulose, 3% methyl cellulose was withdrawn into and infused out of the FEP tube with a syringe. Thereafter the process is repeated with E3 buffer to create a thin layer of methyl cellulose on the inner walls of the tube. Now the fish larva with 200mg/l Tricane in E3 is sucked into the FEP tube and can be exposed to the magnetic field. Methyl cellulose prevents it from sticking to the tube and allows the fish to move smoothly inside the tube.