Electroporation of rhodamine B and acridine orange. (A) Optimisation of the modulation frequency of electroporation. The electroporation was performed at 15 V; 5000 ms; 1 pulse using variable frequencies. Relative fluorescence indicates fold fluorescein signal measured compared to non-electroporated, but otherwise equally treated reference embryos. For extension to other small molecules, the embryos were incubated with 10 ng/ml rhodamine B (B-D) and 1 μg/ml acridine orange (E-G) at stage 2a (B,E), stage 17 (C,F) and stage 36 (D,G) for 40 minutes at 27°C. Electroporation was performed using 330 Hz; 15 V; 5000 ms; 1 pulse. The dotted lines demarcate the outlines of: (B,E) yolk and blastoderm, and (C,D,F,G) yolk and embryo. Embryos are shown in lateral view. Scale bar 250 μM.