Table 1.

Imaging techniques.

Technique	Type of imaging	Applications	Major benefits	Drawbacks	Notable uses	Reference(s)
(SBF-SEM) Serial block face—scanning electron microscopy	Destructive scanning electron microscopy (serial ultramicrotome sectioning)	Studying sub-cellular structures and cell-cell interactions in 3D	High resolution (5–10 nm) through 3D volume	Destroys the sample, feasible only for a relatively small total volume (slow acquisition and large data sets)	Characterization of axon diameter and myelination status in transected mouse optic nerve [5 nm (X–Y), 50 nm (Z), total volume = 50 μm (X–Y), 100 μm (Z)]; 3D-reconstruction of normal and cuprizone-demyelinated axons in mouse corpus callosum [1 nm (X–Y), 80 nm (Z), area per “slice” = 204.54 μm × 61.36 μm]	Giacci et al. (2018) and Fischbach et al. (2019)
(FIB-SEM) Focussed ion beam—scanning electron microscopy	Destructive scanning electron microscopy (focussed ion beam ablation of block face)	Studying sub-cellular structures and cell-cell interactions in 3D	Better Z-resolution than SBF-SEM (<10 nm) due to different surface ablation method	Destroys the sample, typically smaller total volumes can be imaged than for SBF-SEM	3D reconstruction of organelles in a myelinating oligodendrocyte within mouse optic nerve [7.5 nm resolution (X–Y), 30 nm (Z), total volume = 7.72 μm (X), 5.79 μm (Y), 3.81 μm (Z)].	Schertel et al. (2013)
(CLEM) Correlative light and electron microscopy	Correlated light and electron microscopy on the same samples	EM-resolution of samples (cells and sub-cellular structures) with wider contextual information	Places nm-scale EM resolution within the wider context of cells and tissues, allows for easier localization of nano-scale structures by first identifying them through fluorescent microscopy	Requires multiple types of imaging and careful alignment of separate imaging data sets of the same sample	Combined in vivo multiphoton microscopy, confocal microscopy, and FIB-SEM on the same samples of mouse brain (using myelinated axons as landmarks)	Luckner et al. (2018)
(LSFM) Lightsheet fluorescence microscopy	Volumetric fluroescent microscopy (sheet of illuminating light)	Whole organism/organ imaging and tissue architecture	Non-destructive fluorescent imaging of whole animals, organs or tissues	Relatively low resolution (microns)	Mapping expansion and migration of grafted human neural progenitor cells in mouse (whole mouse brain); identification of functionally, morphologically and spatially distinct subtypes of OPC in zebrafish spinal cord (entire zebrafish larvae)	Vogel et al. (2019) and Marisca et al. (2020)
Expansion microscopy	Fluorescent imaging of artificially expanded samples to increase resolution	Imaging organelles, synapses, synaptic vesicles and cell-cell interactions	High resolution beyond capabilities of typical light microscopy objectives, due to expansion of sample (effectively tens of nm)	Lengthy expansion procedure; possible distortion of tissue architecture	Super-resolution visualization of myelinated axons in mouse hippocampus (150-μm thick slices of mouse brain)	Min et al. (2020)
Super-resolution microscopy (e.g., STED—Stimulated Emission Depletion Microscopy)	Super-resolution microscopy	Imaging organelles, synapses, synaptic vesicles and cell-cell interactions	High resolution light microscopy, achieved by limiting or negating the inherent diffraction of light	Limited volumetric information, most super-resolution techniques require post-processing of images (although not STED)	Demonstration of preferential interaction of OPCs with the nodes of Ranvier of large diameter axons in mouse CNS	Serwanski et al. (2017)
Synchrotron X-ray microtomography	Non-destructive volumetric imaging	3D imaging organs and tissues	Better resolution and contrast compared to conventional μCT	High energy X-rays means cannot image live specimens, feasible only for smaller total volumes of dissected tissue (mm-scale)	Visualization of myelinated axons and other structures in a subvolume of mouse neocortex (voxel size of approx. 1 μm3, mm-sized piece of tissue)	Dyer et al. (2017)
Multiphoton	Non-invasive, in vivo fluorescent imaging	Longitudinal or time-lapse studies in live animals or cultured organs/tissues	Live imaging and ability to image cells and structures at depth within tissues, limited photobleaching, can perform optical sectioning	Relatively slow acquisition, resolution is not superior to conventional confocal microscopy	Imaging of OPC migration from SVZ to cortex in postnatal mouse brain slices	Gadea et al. (2009)