Journal Club: Detailed protocols on image acquisition and analysis of mammalian cochleae with phase-contrast X-ray tomography

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Today's journal article

Schaeper JJ, Kampshoff CA, Wolf BJ, Roos L, Michanski S, Ruhwedel T, Eckermann M, Meyer A, Jeschke M, Wichmann C, Moser T, Salditt T. 3D virtual histology of rodent and primate cochleae with multi-scale phase-contrast X-ray tomography. 

Why I picked this article

The inner ear is tiny and delicate, which makes 3D anatomy hard to study without cutting tissue. X‑ray phase‑contrast tomography (XPCT) can image soft tissues that are nearly invisible in standard micro‑CT. This paper is useful because it does not just show images; it benchmarks practical setups (synchrotron and in‑house), sample prep options, phase‑retrieval methods, and achievable voxel sizes from whole‑cochlea overview scans down to ~50 nm region‑of‑interest (ROI) scans of the organ of Corti.

Some of the research findings

  • Species: common marmoset (Callithrix jacchus), mouse, gerbil, Wistar rat; plus a marmoset head with a lifetime electrical cochlear implant (eCI) and rat heads with optical or electrical CI.
  • Fixation/decalcification (typical): 4% paraformaldehyde (PFA); 10% EDTA decalcification 13–18 weeks (marmoset temporal bones). 
  • Some specimens were cleared with iDISCO and embedded in dibenzyl ether (DBE); others were immersed in 100% methanol; many were mounted in agarose in Eppendorf cups. 
  • Histology‑style staining/embedding (for sub‑cellular contrast): mouse apical turns stained with 1% OsO4 (1 h) + 1% uranyl acetate (1 h) or 2% OsO4 (2 h), then EPON embedding cast as 1‑mm cylinders (vacuum compatible and radiation stable).
  • Data are available at: https://data.goettingen-research-online.de/    https://doi.org/10.25625/TU53LS 
Image acquisition and analysis: 
  • DESY PETRA III P10 — GINIX endstation (Hamburg)
    • SR‑CB1 (holographic cone‑beam)
    • SR‑CB2 (wide‑field cone‑beam)
  • ESRF ID16A — nano‑imaging beamline (Grenoble): SR‑CB3: 17.1 keV (option for 33.6 keV)
  • ESRF ID17 — biomedical white‑beam (SR‑PB2)
  • micro‑CT (Göttingen)
    • μCT‑1 (liquid‑metal jet source, Excillum D2; Ga Kα 9.25 keV)
    • μCT‑2 (RX Solutions EasyTOM): switchable micro‑/nano‑focus sources
  • Software: There is a whole table in the article with where to find tools/codes! 
    • HoloTomoToolbox for phase retrieval
    • ASTRA‑Toolbox (FDK) for filtered back‑projection for reconstructions
    • Nabu (ESRF) for ID16A multi‑distance CTF
    • NRStitcher for tiling
    • NVIDIA IndeX and Siemens Cinematic Anatomy for rendering
    • arivis blob‑finder for spiral ganglion neuron segmentation.
From Figure 3C. ZPCT of osmium stained cochlea. Schaeper et al. 

Haruna's takeaway

This is a great methodology paper, where they summarise and compare different equipment and techniques, with parameters openly available for comparison. Very beautiful images shown in this publication give good examples of what could be achieved by using these techniques and optimised protocols. Execution is always hard; we do microCT, but it's all about sample preparation, and given how biological samples always differ from one another, parameter optimisation and mounting strategy for each sample are very important. Looking at this publication kind of makes us want to do more CT scans! 

The resolution of the phase-contrast microCT shown here is very cool and very exciting. 


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This is Haruna's 67/100 of the 100-day challenge to post a science blog article every day! I love inner ear biology & cochlear physiology.