This is IMAGE #2 of a group of neurons from the optic tectum of an albino Xenopus laevis tadpole CNS, stage 47. This neuron cluster is part of an image group. The cells were imaged daily over 2 days and the images show the development of the dendritic arbors of the neurons. IMAGE #1 is a different view of this same time point. The cells were 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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