Time-lapse imaging was performed with a Zeiss LSM710 or Leica SP8 confocal microscope with a ×40, numerical aperture 1.3 inverted oil lens, a 488-nm argon laser, and a 568-nm green HeNe laser. The basal focal plane, which is about 1 μm beneath the basal surface, was selected during live imaging to maximize the basal Myo-II intensity. For the dynamics of ROCK–GFP, MBS–GFP, MBS-RFP, or Flw-YFP, the similar basal focal plane was selected to maximize the basal signal intensity, as basal MyoII dynamic imaging did. The same microscope setup was used when comparing intensity between different samples. To view the signals at different focal planes, images were taken at different Z-stack layers from the basal surface to the apical side.
FRET images of live cultured egg chambers were acquired with a Zeiss LSM710 microscope, by using a similar version of the protocol described in ref. 38 (link). A 458 nm laser was used to excite the sample. CFP and YFP emission signals were collected through channel I (470–510 nm) and channel II (525–600 nm), respectively. To capture single, high-resolution, and stationary images, a ×40/1.3 oil inverted objective was used. CFP and YFP images were acquired simultaneously for most of the experiments. Sequential acquisition of CFP and YFP channels in alternative orders were tested and gave the same result as simultaneous acquisition.
FRET images of live cultured egg chambers were acquired with a Zeiss LSM710 microscope, by using a similar version of the protocol described in ref. 38 (link). A 458 nm laser was used to excite the sample. CFP and YFP emission signals were collected through channel I (470–510 nm) and channel II (525–600 nm), respectively. To capture single, high-resolution, and stationary images, a ×40/1.3 oil inverted objective was used. CFP and YFP images were acquired simultaneously for most of the experiments. Sequential acquisition of CFP and YFP channels in alternative orders were tested and gave the same result as simultaneous acquisition.
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