In our studies of polarity establishment in C. elegans embryos, in order to successfully pierce the chitinous shell and vitelline membrane while minimizing damage to the embryo, the tip of an injection pipette must be both strong and small. To accomplish this, we fabricate quartz reinforced carbon nanopipettes (CNPs). Quartz capillaries were pulled to a tip of 174 ± 59 nm and then reinforced with an interior layer of carbon. Figure 1 (a) summarizes the protocol steps for creating these CNPs. These CNPs were used previously to inject dyes and secondary messengers into epithelial cells, as well as to measure cell membrane potential. They allow for the direct injection of small molecules with minimal damage to, and maximal temporal control over the embryos. In addition, un-reinforced quartz capillaries were pulled to obtain a smooth ~15 µm tip to serve as ‘holding’ pipettes to maintain an embryo’s position during injection, as well as to allow withdrawal of the CNP after drug delivery, as presented in Fig. 1 (c) and (d). Both CNPs and holding pipettes can be fabricated with high reproducibility with in batches (~80 for the CNPs, no limit for the holding pipettes).
Carbon NanoPipette (CNP) fabrication and experimental configuration.
(a) Fabrication procedure of the CNPs. Quartz capillaries are filled with Fe(NO3)3 catalyst and left to dry, then pulled into pipettes of desired geometry. Carbon is grown within the pipette using chemical vapor deposition. (b) SEM images of the pipettes before and after carbon deposition. Scale bars are 100 nm. (c-d) Sketch and microscopy images of the experimental configuration: a quartz holding pipette is used to immobilize the embryo during injection and light suction applied through the holing pipette allows for the retrieval of the CNP. Scale bar in (d) is 10 μm, injection occurs approximately at t = 0:15 (min:sec).