9. Using the micromanipulator, gently move the needle tip laterally, and touch the side of the coverslip (Movie 1). Minimal
contact between the needle tip and the coverslip, while ejecting
liquid from the needle, will suffice to break the needle tip.
Move the needle back and ensure that it was broken by trying
to eject some solution. A bubble (not a stream of solution) is
indicative of a successful needle break. From this point on,
minimize the time that the tip of the needle is not immersed
in oil to prevent clogging.
10. To prepare for embryo injection, slowly move the needle
upwards and replace the needle-breaking slide with the coverslip with glued embryos.
11. Lower the needle slowly until the tip is inside the oil that covers
the embryos. Focus the microscope on the needle tip. Make
sure that the embryo cross-section and the needle tip are in
focus on the same plane by adjusting the position of the needle.
12. Gently push the needle against the vitelline membrane
(Movie 2). The vitelline membrane should display some light
resistance and will eventually be pierced by the pressure from
the needle. For injections in the perivitelline space (e.g.,
membrane-permeable dyes or small molecule inhibitors),
slowly pull back the needle (Movie 2). The vitelline membrane
will retract with the needle, forming a pocket where solution
can be ejected. Membrane-impermeable molecules can be
injected in the yolk before cellularization is complete. Embryos
can then be aged until the appropriate stage (see Note 16). If
the embryo bursts upon injection, reduce the ejection pressure
of the pneumatic pump, use a needle with a thinner tip, or
increase the embryo dehydration time.
13. If embryos need to be moved to an incubation chamber for
aging or to a different microscope for imaging, make sure to
place the slide used to break the needle back on the stage and
lower the needle tip into the oil to prevent needle clogging.
14. If injections were conducted on the same microscope in which
imaging will occur, leave the needle tip inside the oil that covers
the embryos to prevent needle clogging.
3.4 Time-Lapse
Imaging of Collective
Cell Movements
3.4.1 Live Imaging Using
Spinning Disk Confocal
Microscopy
1. Startup Metamorph.
2. If using the Micropoint for laser ablation, make sure that the
laser is calibrated with the objective lens required for ablation
(see Note 17).
3. Place the coverslip containing the embryos on the microscope
stage, and find an embryo using the 10 or 20Â objective and
bright field illumination.
Live Imaging in Drosophila Embryos
209
contact between the needle tip and the coverslip, while ejecting
liquid from the needle, will suffice to break the needle tip.
Move the needle back and ensure that it was broken by trying
to eject some solution. A bubble (not a stream of solution) is
indicative of a successful needle break. From this point on,
minimize the time that the tip of the needle is not immersed
in oil to prevent clogging.
10. To prepare for embryo injection, slowly move the needle
upwards and replace the needle-breaking slide with the coverslip with glued embryos.
11. Lower the needle slowly until the tip is inside the oil that covers
the embryos. Focus the microscope on the needle tip. Make
sure that the embryo cross-section and the needle tip are in
focus on the same plane by adjusting the position of the needle.
12. Gently push the needle against the vitelline membrane
(Movie 2). The vitelline membrane should display some light
resistance and will eventually be pierced by the pressure from
the needle. For injections in the perivitelline space (e.g.,
membrane-permeable dyes or small molecule inhibitors),
slowly pull back the needle (Movie 2). The vitelline membrane
will retract with the needle, forming a pocket where solution
can be ejected. Membrane-impermeable molecules can be
injected in the yolk before cellularization is complete. Embryos
can then be aged until the appropriate stage (see Note 16). If
the embryo bursts upon injection, reduce the ejection pressure
of the pneumatic pump, use a needle with a thinner tip, or
increase the embryo dehydration time.
13. If embryos need to be moved to an incubation chamber for
aging or to a different microscope for imaging, make sure to
place the slide used to break the needle back on the stage and
lower the needle tip into the oil to prevent needle clogging.
14. If injections were conducted on the same microscope in which
imaging will occur, leave the needle tip inside the oil that covers
the embryos to prevent needle clogging.
3.4 Time-Lapse
Imaging of Collective
Cell Movements
3.4.1 Live Imaging Using
Spinning Disk Confocal
Microscopy
1. Startup Metamorph.
2. If using the Micropoint for laser ablation, make sure that the
laser is calibrated with the objective lens required for ablation
(see Note 17).
3. Place the coverslip containing the embryos on the microscope
stage, and find an embryo using the 10 or 20Â objective and
bright field illumination.
Live Imaging in Drosophila Embryos
209