Confocal Microscopy for Axon Diameter

Fluorescence confocal microscopy and image stack capturing were carried out as described in previous publications from our laboratory [27 (link),29 (link),30 (link),32 (link)]. In the present study, we used an Andor Revolution WD spinning disk confocal system (Oxford Instruments, Abingdon-on-Thames, UK) based on a Nikon TiE inverted microscope (60× CFI Plan Apo VC water immersion objective and numerical aperture at 1.40). We captured Z-stack images (8-bit TIFF files) at ~0.10 µm steps for the corpus callosum (Bregma between −1.82 and −2.30 mm) and cortical layer VI (Bregma between +1.98 and +1.78 mm). Image stacks were analyzed with NIH ImageJ. Axon diameters were measured on the Z-projection after Dynamic Reslice. The diameter values from the thickest and thinnest portions in the same axonal segment were compared and plotted.

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Ma D., Deng B., Sun C., McComb D.W, & Gu C. (2022). The Mechanical Microenvironment Regulates Axon Diameters Visualized by Cryo-Electron Tomography. Cells, 11(16), 2533.

Publication 2022

CFI Plan Apo VC

Axonal Corpus callosum Cortical Fluorescence microscopy Immersion Microscope

Corresponding Organization : The Ohio State University

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Variable analysis

independent variables

Fluorescence confocal microscopy and image stack capturing

dependent variables

Axon diameters measured on the Z-projection after Dynamic Reslice
Diameter values from the thickest and thinnest portions in the same axonal segment

control variables

Corpus callosum (Bregma between −1.82 and −2.30 mm)
Cortical layer VI (Bregma between +1.98 and +1.78 mm)
Andor Revolution WD spinning disk confocal system (Oxford Instruments, Abingdon-on-Thames, UK) based on a Nikon TiE inverted microscope (60× CFI Plan Apo VC water immersion objective and numerical aperture at 1.40)
Z-stack images (8-bit TIFF files) captured at ~0.10 µm steps

controls

Previous publications from our laboratory [27, 29, 30, 32] are used as positive controls for the experimental methods

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