the following transfer and positioning of the embryos. Position
the LMP agarose tube and the plate with embryos under the
stereomicroscope. Using a plastic pipette, transfer 8 embryos
with as little medium as possible (no more than 0.1 mL) into
the LMP agarose tube.
7. Quickly take back the embryos into the transfer pipette with
0.3 mL of LMP agarose and place on top of the agarose mould.
Using the hair knife, position the embryos on the agarose holes
so that the yolk sits into the holes. Orient the embryos either in
a lateral or dorsal position. Let the LMP solidify and the
embryos set for about 10 min.
Alternatively, for short time-lapse movies, it is possible to
mount the embryos in 3% methyl cellulose. Make a shallow agarose
mould, transfer the embryos to an Eppendorf tube containing
0.9 mL 3% methyl cellulose and 0.1 mL of tricaine, and then
transfer the embryos to the agarose mould with little methyl cellulose. Use a hair knife to orient the embryos.
3.2 Time-Lapse
Imaging
1. Place the embryo dish on the motorized stage and add dipping
medium (E3/tricaine) up to the rim of the dish.
2. Select the objective to be used (see Note 3) and focus on one of
the embryos at the edge of the plate. Find the cells of interest.
3. Set up the acquisition protocol in the software, recording the
required fluorescent channels. Always take a similar stack of the
bright field channel that will be required to correct 3D drift of
the movie (see Subheading 3.3). For an overnight time-lapse,
set the lasers power and exposure time to the minimum levels
to generate a dim but visible signal (see Note 4). In our setup, a
15–20% lasers power, 400ms exposure and 180 gain, with
2 Â 2 binning of the camera.
4. The z step and range of the stack will depend on your specific
experiment. For good resolution movies of migrating trunk
neural crest on lateral view, we use a 2 μm z step and a total
stack of 70 μm (or 140 μm to acquire both sides of the
embryo), leaving some space above and below the region of
interest in case of z drift. For high resolution images, that
would allow volume reconstruction, a 1 μm z step is required,
in which case long-term (>10 h) imaging is not possible due to
photo-bleaching.
The time step and total length of imaging will vary depending on the specific question (see Note 5). If a motorized stage is
in place, the time interval between images will determine how
many embryos can be imaged simultaneously.
5. Check that all the embryos are in focus and start the acquisition. After 1 h of acquisition, check that all embryos are still in
focus and have not drifted; refocus if at all necessary.
Tools to Study Neural Crest Migration in Zebrafish
91
the LMP agarose tube and the plate with embryos under the
stereomicroscope. Using a plastic pipette, transfer 8 embryos
with as little medium as possible (no more than 0.1 mL) into
the LMP agarose tube.
7. Quickly take back the embryos into the transfer pipette with
0.3 mL of LMP agarose and place on top of the agarose mould.
Using the hair knife, position the embryos on the agarose holes
so that the yolk sits into the holes. Orient the embryos either in
a lateral or dorsal position. Let the LMP solidify and the
embryos set for about 10 min.
Alternatively, for short time-lapse movies, it is possible to
mount the embryos in 3% methyl cellulose. Make a shallow agarose
mould, transfer the embryos to an Eppendorf tube containing
0.9 mL 3% methyl cellulose and 0.1 mL of tricaine, and then
transfer the embryos to the agarose mould with little methyl cellulose. Use a hair knife to orient the embryos.
3.2 Time-Lapse
Imaging
1. Place the embryo dish on the motorized stage and add dipping
medium (E3/tricaine) up to the rim of the dish.
2. Select the objective to be used (see Note 3) and focus on one of
the embryos at the edge of the plate. Find the cells of interest.
3. Set up the acquisition protocol in the software, recording the
required fluorescent channels. Always take a similar stack of the
bright field channel that will be required to correct 3D drift of
the movie (see Subheading 3.3). For an overnight time-lapse,
set the lasers power and exposure time to the minimum levels
to generate a dim but visible signal (see Note 4). In our setup, a
15–20% lasers power, 400ms exposure and 180 gain, with
2 Â 2 binning of the camera.
4. The z step and range of the stack will depend on your specific
experiment. For good resolution movies of migrating trunk
neural crest on lateral view, we use a 2 μm z step and a total
stack of 70 μm (or 140 μm to acquire both sides of the
embryo), leaving some space above and below the region of
interest in case of z drift. For high resolution images, that
would allow volume reconstruction, a 1 μm z step is required,
in which case long-term (>10 h) imaging is not possible due to
photo-bleaching.
The time step and total length of imaging will vary depending on the specific question (see Note 5). If a motorized stage is
in place, the time interval between images will determine how
many embryos can be imaged simultaneously.
5. Check that all the embryos are in focus and start the acquisition. After 1 h of acquisition, check that all embryos are still in
focus and have not drifted; refocus if at all necessary.
Tools to Study Neural Crest Migration in Zebrafish
91