Serial Sectioning and Tomography of Drosophila Larval Brains

Epon-embedded Drosophila larval brains were cut transversally to the ventral nerve cord with Leica Ultracut UCT ultramicrotome. Totally, 200–250 nm thick serial sections were collected on formvar carbon-coated slot grids and 10 nm colloidal gold particles were deposited on both surfaces for fiducials. Samples were imaged on a FEI Tecnai G2 20 operating at 200 kV (Lab6) with a FEI eagle 2k CCD camera at a nominal magnification of 14,500 that resulted in a resolution of 1.5 nm per pixel. FEI single tilt tomography holder was tilted over a range of ±65° according to a Saxton’s scheme (2° starting angle, for a total of 87 images collected) using the FEI Xplore3d acquisition software. Tilted images alignment, tomography reconstruction (WBP) and tomograms joining was done with the IMOD software package^{52 (link)}. ER structures were rendered by manually segmenting the membranes of ER profiles using IMOD software^{53 (link)}.

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Espadas J., Pendin D., Bocanegra R., Escalada A., Misticoni G., Trevisan T., Velasco del Olmo A., Montagna A., Bova S., Ibarra B., Kuzmin P.I., Bashkirov P.V., Shnyrova A.V., Frolov V.A, & Daga A. (2019). Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon. Nature Communications, 10, 5327.

Publication 2019

Ultracut UCT ultramicrotome Tecnai G2 20 eagle 2k CCD camera Xplore3d acquisition software

Brains Carbon Colloidal gold Cord Drosophila Eagle Epon Formvar Imod Larval Membranes Nerve Reconstruction Tomograms Ultramicrotome

Corresponding Organization : Ikerbasque

Other organizations : University of the Basque Country, Biofisika, IRCCS Eugenio Medea, IMDEA Nanoscience, University of Padua, Frumkin Institute of Physical Chemistry and Electrochemistry, Research Institute of Physical Chemical Medicine

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Variable analysis

independent variables

Ultramicrotome used to cut the Epon-embedded Drosophila larval brains transversally to the ventral nerve cord
Thickness of the serial sections (200-250 nm)
Deposition of 10 nm colloidal gold particles on both surfaces of the sections for fiducials
Microscope used (FEI Tecnai G2 20) operating at 200 kV (Lab6) with a FEI eagle 2k CCD camera
Nominal magnification (14,500) resulting in a resolution of 1.5 nm per pixel
Tilt range (±65°) and Saxton's scheme (2° starting angle, for a total of 87 images collected) used for single tilt tomography
Software used for image alignment, tomography reconstruction (WBP), and tomograms joining (IMOD)

dependent variables

Structures of the endoplasmic reticulum (ER) rendered by manually segmenting the membranes of ER profiles using IMOD software

control variables

Formvar carbon-coated slot grids used to collect the serial sections

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