bare eyes when the bright-field light is on. There is no need for
epifluorescence mode (see Note 9).
3. Under bright field, adjust the focus and move the stage slightly
to find the shadow of the unfocused embryo and then focus
properly. Go through the rows of the embryos to find one of
appropriate stages by examining the depth of cellularization.
Further inspect the orientation of the embryo: the embryo
should appear very symmetric including the pole cells at the
posterior end. Damaged embryos can show visible leakage of
yolk droplets. Finally move the focus to the surface of the
embryo to facilitate focusing under confocal mode.
4. Set the laser power to low levels and visualize the embryo in live
mode. Do a final check on the age, orientation, and healthiness
of the embryo. Red fluorescent protein is almost always weaker
and faster bleached than GFP so use 488 nm laser alone for this
purpose if possible (see Note 10).
5. Once the embryo to be imaged is identified, adjust the laser
power, gain, zoom factor, and temporal resolution. Find a
balance between them to minimize photobleaching and maximize the image quality (see Note 11).
4 Notes
1. Bleach can lose potency over time and the dechorionation
becomes slow. It is tempting to push and roll the embryo to
facilitate dechorionation. However, doing so often results in
embryo damage or embryos rolling inside the vitelline membrane. This will lead to difficulties in properly orienting the
embryos when trying to precisely image the mesoderm. Furthermore, embryos rolled inside the vitelline membrane always
roll back to the original position exactly during ventral furrow
formation. Therefore, the cells being imaged will be moving in
or out of the field of view.
2. Usually preparing water drops and paper towels for the washing step is sufficient for dechorionation. While not all embryos
will appear to be dechorionated before washing, they will by
the first two to three rounds of drying and washing cycle. As
the water rushes through the small paper towel from its corner,
all embryos should be completely dechorionated, if they are
not already. When the bleach is not potent enough, drying the
small paper towel and reapplying the bleach can significantly
speed up the dechorionation process.
74
Lingkun Gu and Mo Weng
epifluorescence mode (see Note 9).
3. Under bright field, adjust the focus and move the stage slightly
to find the shadow of the unfocused embryo and then focus
properly. Go through the rows of the embryos to find one of
appropriate stages by examining the depth of cellularization.
Further inspect the orientation of the embryo: the embryo
should appear very symmetric including the pole cells at the
posterior end. Damaged embryos can show visible leakage of
yolk droplets. Finally move the focus to the surface of the
embryo to facilitate focusing under confocal mode.
4. Set the laser power to low levels and visualize the embryo in live
mode. Do a final check on the age, orientation, and healthiness
of the embryo. Red fluorescent protein is almost always weaker
and faster bleached than GFP so use 488 nm laser alone for this
purpose if possible (see Note 10).
5. Once the embryo to be imaged is identified, adjust the laser
power, gain, zoom factor, and temporal resolution. Find a
balance between them to minimize photobleaching and maximize the image quality (see Note 11).
4 Notes
1. Bleach can lose potency over time and the dechorionation
becomes slow. It is tempting to push and roll the embryo to
facilitate dechorionation. However, doing so often results in
embryo damage or embryos rolling inside the vitelline membrane. This will lead to difficulties in properly orienting the
embryos when trying to precisely image the mesoderm. Furthermore, embryos rolled inside the vitelline membrane always
roll back to the original position exactly during ventral furrow
formation. Therefore, the cells being imaged will be moving in
or out of the field of view.
2. Usually preparing water drops and paper towels for the washing step is sufficient for dechorionation. While not all embryos
will appear to be dechorionated before washing, they will by
the first two to three rounds of drying and washing cycle. As
the water rushes through the small paper towel from its corner,
all embryos should be completely dechorionated, if they are
not already. When the bleach is not potent enough, drying the
small paper towel and reapplying the bleach can significantly
speed up the dechorionation process.
74
Lingkun Gu and Mo Weng