Size and specimen-dependent strategy for x-ray micro-ct and tem correlative analysis of nervous system samples

Abstract

Correlative approaches are a powerful tool in the investigation of biological samples, but require specific preparation procedures to maintain the strength of the employed methods. Here we report the optimization of the embedding protocol of nervous system samples for a correlative synchrotron X-ray computed microtomography (micro-CT) and transmission electron microscopy (TEM) approach. We demonstrate that it is possible to locate, with the micrometric resolution of micro-CT, specific volumes of interest for a further ultrastructural characterization to be performed with TEM. This approach can be applied to samples of different size and morphology up to several cm. Our optimized method represents an invaluable tool for investigating those pathologies in which microscopic alterations are localized in few confined regions, rather than diffused in entire tissues, organs or systems. We present a proof of concept of our method in a mouse model of Globoid Cells Leukodistrophy.

Characterisation of cuticular inflation development and ultrastructure in Trichuris muris using correlative X-ray computed tomography and electron microscopy

“…Here we present correlative tomography as an approach which addresses these issues and allows for efficient imaging of multiple inflations within the same worm. X-ray computed tomography (CT) was used to non-destructively generate a 3D image of the interior sample microstructure, which allowed the steering of subsequent milling and sectioning to a desired region of interest for further electron microscopy [18][19][20] . Combining this approach with Serial Blockface Scanning Electron Microscopy (SBF-SEM), in which the sample is serially sectioned and imaged, allowed the acquisition of 3D datasets over multiple regions of interest in one sample.…”

Optimising complementary soft tissue synchrotron X-ray microtomography for reversibly-stained central nervous system samples

“…Osmium (which binds lipids) demonstrated the greatest improvement in tissue contrast (Fig. 3D ), but this was restricted to the white matter perimeter by peripherally-deposited osmium forming a barrier that prevents osmium penetration to deeper tissue 23 , 34 . LI showed the greatest potential of the four stains tested.…”

“…If more physically-stable samples are required such as for repeat imaging or long duration imaging at high magnification (thinner, less efficient scintillators must be used to achieve high resolution with high magnification objectives), epoxy resin offered ultimate sample stability. The resin formula we utilised was adapted from EM protocols 40 so EM could theoretically follow SRμCT, as previously demonstrated 23 . However, resins preclude the use of tissue for most subsequent histology techniques and if these were required, wax embedding would be the method of choice.…”

“…Within SRμCT studies, the number of collected projections reported can vary widely. Various nervous system studies have collected 720 20 , 21 , 501–2000 22 , 1600 2 , 1800–2400 23 and 2000–2400 3 projections. The minimal number of projections that should be collected to avoid undersampling at the edge of the field of view is calculated by multiplying the pixel width of an imaging system (perpendicular to the rotation axis) by π/2 19 .…”

Multiscale imaging of the rat brain using an integrated diceCT and histology workflow

“…X-ray imaging of non-mineralized soft tissues is a signi cant challenge for neuroanatomical studies, causing the brain to be intrinsically di cult to document through such means. Contrast agents, like iodine, potentially address this issue by enabling the visualization of soft neuroanatomical features at high resolutions (Wong et al 2013;Lautenschlager et al 2014;Gignac et al 2016;Parlanti et al, 2017;Naumann and Olsson 2018). However, neural tissues' resilience to multi-faceted imaging pipelines has not previously been quanti ed nor tested explicitly.…”