4 Notes
1. Water immersion and long distance lenses are required. The
best compromise between magnification and numerical aperture needs to be found. Numerical apertures below 1.0 are not
ideal as they will require longer exposure and higher lens
power, which will produce photo damage. If using a 40Â
lens, we advise to take 2–3 adjacent fields of view and stitch
them together, ensuring that the cells of interest do not go out
of plane during the experiment. Also, place an open container
with water next to the microscope stage to prevent
evaporation.
2. Regular commercial plastic petri non-cell culture dishes of any
size can be used. We routinely use 60 mm and 35 mm, but the
size of the dish will depend on the microscope inset and the
lens size. If using a motorized stage to make several movies in
one session, the lens has to be able to freely travel from one
embryo to the next without touching the walls of the dish.
Small dishes are more suitable when treating embryos with
drugs as smaller amounts are required. In which case it is
important to add drug to the agarose mould.
3. Bubbles can form underneath the plastic cast. To avoid that,
spray the cast with 70% ethanol before positioning it on top of
the agarose. After the agarose hardens, leave it to air dry for
20 min to evaporate any alcohol.
4. It is important that the fluorescent protein to be imaged is
expressed at good levels before starting to image. We have
observed that imaging cells with low fluorescent levels by
increasing laser power quickly bleaches the initial protein present and cells are not able to accumulate it thereafter, generating
a black movie.
5. The time step has to be adapted to the question at hand. For
example, to study actin flow in neural crest, the faster the
acquisition time the better it is, although a 2 min interval is
generally good. To study neural crest differentiation, the acquisition time can be set to 30 min or 1 h intervals, which allows
for a longer total time of the movie.
6. The type of files obtained will depend on the software of the
system used. Fiji will read most of the common microscopy
software directly. If yours is not directly read, export your
movies as TIFF series with maximal resolution. Open them in
Fiji clicking “File > Import > Image sequence,” a Sequence
Option window will open. Indicate the total number of images,
starting image and increment, and whether to sort the images
numerically. For example, if your channels are one after the
other and you have 30 z steps and ten time points, then for the
Tools to Study Neural Crest Migration in Zebrafish
103
1. Water immersion and long distance lenses are required. The
best compromise between magnification and numerical aperture needs to be found. Numerical apertures below 1.0 are not
ideal as they will require longer exposure and higher lens
power, which will produce photo damage. If using a 40Â
lens, we advise to take 2–3 adjacent fields of view and stitch
them together, ensuring that the cells of interest do not go out
of plane during the experiment. Also, place an open container
with water next to the microscope stage to prevent
evaporation.
2. Regular commercial plastic petri non-cell culture dishes of any
size can be used. We routinely use 60 mm and 35 mm, but the
size of the dish will depend on the microscope inset and the
lens size. If using a motorized stage to make several movies in
one session, the lens has to be able to freely travel from one
embryo to the next without touching the walls of the dish.
Small dishes are more suitable when treating embryos with
drugs as smaller amounts are required. In which case it is
important to add drug to the agarose mould.
3. Bubbles can form underneath the plastic cast. To avoid that,
spray the cast with 70% ethanol before positioning it on top of
the agarose. After the agarose hardens, leave it to air dry for
20 min to evaporate any alcohol.
4. It is important that the fluorescent protein to be imaged is
expressed at good levels before starting to image. We have
observed that imaging cells with low fluorescent levels by
increasing laser power quickly bleaches the initial protein present and cells are not able to accumulate it thereafter, generating
a black movie.
5. The time step has to be adapted to the question at hand. For
example, to study actin flow in neural crest, the faster the
acquisition time the better it is, although a 2 min interval is
generally good. To study neural crest differentiation, the acquisition time can be set to 30 min or 1 h intervals, which allows
for a longer total time of the movie.
6. The type of files obtained will depend on the software of the
system used. Fiji will read most of the common microscopy
software directly. If yours is not directly read, export your
movies as TIFF series with maximal resolution. Open them in
Fiji clicking “File > Import > Image sequence,” a Sequence
Option window will open. Indicate the total number of images,
starting image and increment, and whether to sort the images
numerically. For example, if your channels are one after the
other and you have 30 z steps and ten time points, then for the
Tools to Study Neural Crest Migration in Zebrafish
103