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of the FEP tubes with methyl cellulose, 3% methyl cellulose was withdrawn into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.