Imaging Embryonic Morphogenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 384

ability of a standard ablation laser tuned to 440 nm using Coumarin dye to
perforate the eggshell.
iii. Rinse embryos three times with embryonic growth medium (EGM; for a
detailed recipe, see Shelton and Bowerman, 1996 )+3 m g/mL Nile Blue A
(Sigma) + 1-2 m g/mL cytochalasin D or nocodazole. The addition of Nile
blue allows the assessment of perforation of the eggshell; permeabilized
embryos will take up the dye into granules in gut cells.
iv. Cover with a 30 m L drop of EGM plus drug. Stock solutions of 2 mg/mL
cytochalasin D or nocodazole (Sigma) in DMSO can be stored at 4 C.
v. Pipette a ring of silicon oil around the drop, and four dots of silicon
vacuum grease (Dow Corning) to the corners of the coverslip. It is typically
convenient to insert vacuum grease into a 10 mL plastic syringe without
needle, from which it can then be extruded. The grease ''feet'' provide a
removable spacer that allows a microscope slide to be affixed to the
coverslip.
vi. Invert a slide over the coverslip to form the mount. Press gently to allow fluid
to contact the slide.
2. Perforating the eggshell

i. Determine the position of the ablation beam by moving to a region of the
mount away from the embryos. Focus on the coverslip, and crack the
coverslip using the laser. In the case of the standard manual Micropoint
laser, a sliding neutral density grating can be used to attenuate the beam
strength. Beam amplitude should be just sufficient to crack the coverslip.
If more power is desired later, then the grating can be adjusted

accordingly.
ii. Select an embryo of the desired developmental stage. Find a region of the
embryo where there is a space between the cells and the eggshell.
Embryos are typically oriented within the eggshell such that there is a
larger space between the anterior end of the embryo and the eggshell than
in other regions of the embryo, making this region convenient for laser
irradiation.
iii. Position the embryo so that the eggshell of this region is over where the
ablation laser will hit.
iv. Using a foot pedal or push button, pulse the laser. Usually only one hit is
necessary, but sometimes more pulses are required.
v. It is often possible to tell that the eggshell is perforated because the embryo
will move slightly toward the perforation site. However, we have found that
sufficiently small holes will not induce this response, yet dye penetration
will nevertheless be observed.
vi. If needed, additional perforations can be induced to increase the rate of
penetration of the compound of interest. However, we have found that the
number of viable embryos obtained in these cases goes down markedly.