This is IMAGE #4 (day 3) of a developing neuron from the optic tectum of an albino Xenopus laevis tadpole CNS, stage 47. The cell was imaged daily over 3 days and shows the development of the dendritic arbor and axon over time. This is a lower zoom image of same time point shown in IMAGE #3. The cell was transfected with in vivo electroporation of plasmid DNA encoding eGFP ~24 hours before the image was acquired. The intact, living tadpole was anesthetized, positioned under a glass coverslip and this image was taken directly through its transparent skin using a custom built two-photon microscope.
Specifications of the custom built two-photon microscope, including the laser sources, signal amplification, PMT specifications, and YFP filter sets are described in: Ruthazer ES, Li J, Cline HT. 2006. Stabilization of axon branch dynamics by synaptic maturation. J Neurosci 26(13):3594-3603. The methods of cell transfection with electroporation can be found here: Bestman JE, Ewald RC, Chiu S-L, Cline HT. 2006. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protocols 1(3):1267-1272. Further explanation can be found here: Bestman JE, Cline HT. 2008. The RNA binding protein CPEB regulates dendrite morphogenesis and neuronal circuit assembly in vivo. Proc Natl Acad Sci U S A 105(51):20494-20499. Bestman JE, Cline HT. 2009. The Relationship between Dendritic Branch Dynamics and CPEB-Labeled RNP Granules Captured in Vivo. Front Neural Circuits 3:10.
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