15. Needles can be prepared for microinjection using a needle
puller. It is important to use needles with thin, long tips that
do not break upon contact with the embryo. For the Sutter
Instrument Flaming/Brown Micropipette Puller, we use the
following parameters: pressure 250, heat 492, pull 110, velocity
110, and delay 200. A longer tip can be obtained by reducing
the temperature of the filament, by pulling more slowly, or by
increasing the pressure value.
16. To age embryos, use a 100 mm Petri dish as an incubation
chamber. Place two strips of plasticine in the dish to be used as
supports for the coverslip (Fig. 2a). Arrange wet, fine paper
towels around the circumference of the dish to prevent dehydration. Cover the Petri dish and incubate for the time necessary (see Note 8).
17. To calibrate the Micropoint, place the mirror slide on the
microscope stage, and focus on the slide using the objective
lens that needs to be calibrated (do not forget to add oil on the
objective if it is an oil immersion lens). Find an area of the
mirror slide with no scratches or previous calibration patterns.
Use the calibration routine provided by the software to ensure
accurate laser targeting with the objective lens to be used for
ablation. The Micropoint calibration routine in Metamorph
fires the laser in a 3 Â 3 grid. The software tries to automatically
detect the point where the laser hit the mirror slide based on
the change in contrast between images acquired before and
after the laser is fired. Both the exposure time used to collect
the images in the calibration routine and the laser power used
to fire the laser will determine the success of the automated
spot detection. Aim to generate small spots, so that their
position is well defined, by using short exposure times (e.g.,
50 ms with bright field illumination) and low Micropoint powers. The Micropoint laser power can be controlled
with a neutral density filter that has 30 positions (we typically
set the neutral density filter to positions 20–25, with position
30 being that of minimal attenuation), and with a motorized
attenuator controlled from the software (we often use transmittance values of 5–30% for calibration). If the automated
routine fails, it is possible to manually calibrate the Micropoint
by clicking on the spots generated by the laser on the mirror
slide during the calibration routine. When calibration is complete, a red rectangle will be displayed on the screen, indicating
the region where the laser is properly calibrated. If a trapezoid
rather than a rectangle is displayed, the calibration has not
worked and should be repeated.
18. As the lens approaches the coverslip, transition from the coarse
to the fine focus adjustment controls on the microscope to
avoid hitting the coverslip with the objective.
Live Imaging in Drosophila Embryos
215
puller. It is important to use needles with thin, long tips that
do not break upon contact with the embryo. For the Sutter
Instrument Flaming/Brown Micropipette Puller, we use the
following parameters: pressure 250, heat 492, pull 110, velocity
110, and delay 200. A longer tip can be obtained by reducing
the temperature of the filament, by pulling more slowly, or by
increasing the pressure value.
16. To age embryos, use a 100 mm Petri dish as an incubation
chamber. Place two strips of plasticine in the dish to be used as
supports for the coverslip (Fig. 2a). Arrange wet, fine paper
towels around the circumference of the dish to prevent dehydration. Cover the Petri dish and incubate for the time necessary (see Note 8).
17. To calibrate the Micropoint, place the mirror slide on the
microscope stage, and focus on the slide using the objective
lens that needs to be calibrated (do not forget to add oil on the
objective if it is an oil immersion lens). Find an area of the
mirror slide with no scratches or previous calibration patterns.
Use the calibration routine provided by the software to ensure
accurate laser targeting with the objective lens to be used for
ablation. The Micropoint calibration routine in Metamorph
fires the laser in a 3 Â 3 grid. The software tries to automatically
detect the point where the laser hit the mirror slide based on
the change in contrast between images acquired before and
after the laser is fired. Both the exposure time used to collect
the images in the calibration routine and the laser power used
to fire the laser will determine the success of the automated
spot detection. Aim to generate small spots, so that their
position is well defined, by using short exposure times (e.g.,
50 ms with bright field illumination) and low Micropoint powers. The Micropoint laser power can be controlled
with a neutral density filter that has 30 positions (we typically
set the neutral density filter to positions 20–25, with position
30 being that of minimal attenuation), and with a motorized
attenuator controlled from the software (we often use transmittance values of 5–30% for calibration). If the automated
routine fails, it is possible to manually calibrate the Micropoint
by clicking on the spots generated by the laser on the mirror
slide during the calibration routine. When calibration is complete, a red rectangle will be displayed on the screen, indicating
the region where the laser is properly calibrated. If a trapezoid
rather than a rectangle is displayed, the calibration has not
worked and should be repeated.
18. As the lens approaches the coverslip, transition from the coarse
to the fine focus adjustment controls on the microscope to
avoid hitting the coverslip with the objective.
Live Imaging in Drosophila Embryos
215