Inside neuronal railways in in vitro cell culture

Beyond their known conventional functions in supporting cellular architecture and shape, microtubules act also as railways to orchestrate microtubule-based transport of organelles and proteins and this is particularly relevant in neurons. High-quality imaging, for instance confocal spinning disk, can help to answer still open questions in neuroscience and cytoskeleton fields regarding microtubule 3D organization, how microtubule dynamic and stability are regulated and how these regulatory mechanisms affect cell functions and, more generally, intellectual functions.

Here we report mouse hippocampal neurons obtained from mice pups and processed by immunofluorescence after 12 days of in vitro cell culture assay. In particular, neurons were stained for beta III Tubulin (in magenta), a microtubule element of the tubulin family found almost exclusively in neurons, and Camsap2 (in green), a microtubule-organizing protein that specifically binds the minus-end of non-centrosomal microtubules and regulates their dynamics and organization.

In Figure A we compare widefield acquisition with high-resolution imaging obtained with CrestOptics system (100x oil objective, 1.45 NA), both in the whole field of view and in a specific area of interest. In Figure B we show different elaborations of a 3D high-resolved Z-stack revealing that neuronal dendrites and axon are stained positively for Camsap2 and many individual microtubule-ends were decorated with Camsap2 puncta. We also measured the mean intensity profile along a specific region of interest (highlighted in yellow) and this allows us to appreciate the alternating organization of microtubules and their minus end associated protein.

Figure A

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Figure B

3D views

Maximum Intensity Projection

Mean intensity profile (along the yellow highlighted line)

3D movie

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