Subscribe to RSS
DOI: 10.1055/a-2111-8415
Ex Vivo Micro-CT in Ophthalmology: Preparation and Contrasting for Non-invasive 3D-Visualisation
Article in several languages: English | deutschAuthors

Abstract
X-ray-based micro-computed tomography (micro-CT) is a largely non-destructive imaging method for the visualisation and analysis of internal structures in the ex vivo eye and affords high resolution. In contrast to other high-resolution imaging methods, micro-CT enables spatial recording of larger and more complex tissue structures, such as the anterior chamber of the eye. Special contrasting methods help to enhance the absorption properties of soft tissue, that is otherwise only weakly radiopaque. Critical point drying (CPD), as primarily used in scanning electron microscopy, offers an additional tool for improving differential contrast properties in soft tissue. In the visualisation of intraosseous soft tissue, such as the efferent lacrimal ducts, sample treatment by decalcification with ethylenediaminetetraacetic acid and subsequent CPD provides good results for micro-CT. Micro-CT can be used for a wide range of questions in 1. basic research, 2. application-related studies in ophthalmology (e.g. evaluation of the preclinical application of microstents for glaucoma treatment or analysis of the positioning of intraocular lenses) but also 3. as a supplement to ophthalmological histopathology.
Key words
anterior chamber - iridocorneal angle - lacrimal duct - micro-computed tomography - ocular structures - critical point dryingPublication History
Received: 16 June 2023
Accepted: 06 October 2023
Article published online:
13 December 2023
© 2023. Thieme. All rights reserved.
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
-
References/Literatur
- 1
Gibson E,
Gaed M,
Gómez JA.
et al.
3D prostate histology image reconstruction: Quantifying the impact of tissue deformation
and histology section location. J Pathol Inform 2013; 4: 31
Reference Ris Wihthout Link
- 2
Hann CR,
Bentley MD,
Vercnocke A.
et al.
Imaging the aqueous humor outflow pathway in human eyes by three-dimensional micro-computed
tomography (3D micro-CT). Exp Eye Res 2011; 92: 104-111
Reference Ris Wihthout Link
- 3
Enders C,
Braig EM,
Scherer K.
et al.
Advanced Non-Destructive Ocular Visualization Methods by Improved X-Ray Imaging Techniques.
PLoS One 2017; 12: e0170633
Reference Ris Wihthout Link
- 4
Leszczyński B,
Sojka-Leszczyńska P,
Wojtysiak D.
et al.
Visualization of porcine eye anatomy by X-ray microtomography. Exp Eye Res 2018; 167:
51-55
Reference Ris Wihthout Link
- 5
Tkachev SY,
Mitrin BI,
Karnaukhov NS.
et al.
Visualization of different anatomical parts of the enucleated human eye using X-ray
micro-CT imaging. Exp Eye Res 2021; 203: 108394
Reference Ris Wihthout Link
- 6
Runge J,
Stahnke T,
Guthoff RF.
et al.
Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed
3D-visualization of eye anatomy with special emphasis on critical point drying. Quant
Imaging Med Surg 2022; 12: 4361-4376
Reference Ris Wihthout Link
- 7
Hann CR,
Vercnocke AJ,
Bentley MD.
et al.
Anatomic changes in Schlemmʼs canal and collector channels in normal and primary open-angle
glaucoma eyes using low and high perfusion pressures. Invest Ophthalmol Vis Sci 2014;
55: 5834-5841
Reference Ris Wihthout Link
- 8
Lifton JJ,
Malcolm AA,
McBride JW.
An experimental study on the influence of scatter and beam hardening in x-ray CT for
dimensional metrology. Meas Sci Technol 2016; 27: 015007
Reference Ris Wihthout Link
- 9
Guzman Aparicio MA,
Chen TC.
New views on three-dimensional imaging technologies for glaucoma: An overview. Curr
Opin Ophthalmol 2022; 33: 103-111
Reference Ris Wihthout Link
- 10
Bohn S,
Stahnke T,
Sperlich K.
et al.
In vivo Histology of the Cornea – From the Rostock Cornea Module to the Rostock Electronic
Slit Lamp – A Clinical Proof of Concept Study. Klin Monbl Augenheilkd 2020; 237: 1442-1454
Reference Ris Wihthout Link
- 11
Glarin RK,
Nguyen BN,
Cleary JO.
et al.
MR-EYE: High-Resolution MRI of the Human Eye and Orbit at Ultrahigh Field (7 T). Magn
Reson Imaging Clin N Am 2021; 29: 103-116
Reference Ris Wihthout Link
- 12
Helms RW,
Minhaz AT,
Wilson DL.
et al.
Clinical 3d imaging of the anterior segment with ultrasound biomicroscopy. Transl
Vis Sci Technol 2021; 10: 1-12
Reference Ris Wihthout Link
- 13
Yakovlev MA,
Vanselow DJ,
Ngu MS.
et al.
A wide-field micro-computed tomography detector: micron resolution at half-centimetre
scale. J Synchrotron Radiat 2022; 29: 505-514
Reference Ris Wihthout Link
- 14
Aumann S,
Donner S,
Fischer J.
et al.
Optical Coherence Tomography (OCT): Principle and Technical Realization. In: High
Resolution Imaging in Microscopy and Ophthalmology: New Frontiers in Biomedical Optics
[Internet] Cham: Springer International Publishing; 2019: 59-85
Reference Ris Wihthout Link
- 15
Fouquet C,
Gilles JF,
Heck N.
et al.
Improving axial resolution in confocal microscopy with new high refractive index mounting
media. PLoS One 2015; 10: 1-17
Reference Ris Wihthout Link
- 16
Wang S,
Larina IV.
High-resolution imaging techniques in tissue engineering. In:
Narayan RJ.
ed.
Monitoring and Evaluation of Biomaterials and their Performance in Vivo. Sawston,
Cambridge: Woodhead Publishing; 2017
Reference Ris Wihthout Link
- 17
Niendorf T,
Paul K,
Graessl A.
et al.
Ophthalmologische Bildgebung mit Ultrahochfeld-Magnetresonanztomografie: technische
Innovationen und wegweisende Anwendungen. Klin Monbl Augenheilkd 2014; 231: 1187-1195
Reference Ris Wihthout Link
- 18
Runge J,
Kischkel S,
Keiler J.
et al.
Experimental glaucoma microstent implantation in two animal models and human donor
eyes – an ex vivo micro-CT based evaluation of applicability. Quant Imaging Med Surg
[accepted].
Reference Ris Wihthout Link
- 19
Metscher BD.
Micro CT for comparative morphology: Simple staining methods allow high-contrast 3D
imaging of diverse non-mineralized animal tissues. BMC Physiol 2009; 9: 11
Reference Ris Wihthout Link
- 20
Pauwels E,
Van Loo D,
Cornillie P.
et al.
An exploratory study of contrast agents for soft tissue visualization by means of
high resolution X-ray computed tomography imaging. J Microsc 2013; 250: 21-31
Reference Ris Wihthout Link
- 21
Koç MM,
Aslan N,
Kao AP.
et al.
Evaluation of X-ray tomography contrast agents: A review of production, protocols,
and biological applications. Microsc Res Tech 2019; 82: 812-848
Reference Ris Wihthout Link
- 22
Keiler J,
Richter S,
Wirkner CS.
Evolutionary morphology of the hemolymph vascular system in hermit and king crabs
(Crustacea: Decapoda: Anomala). J Morphol 2013; 274: 759-778
Reference Ris Wihthout Link
- 23
Nordestgaard BG,
Rostgaard J.
Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative
and qualitative study on isolated hepatocytes. J Microsc 1985; 137: 189-207
Reference Ris Wihthout Link
- 24
Horridge GA,
Tamm SL.
Critical Point Drying for Scanning Electron Microscopic Study of Ciliary Motion. Science
1969; 163: 817-818
Reference Ris Wihthout Link
- 25
Jensen OA,
Prause JU,
Laursen H.
Shrinkage in preparatory steps for SEM. A study on rabbit corneal endothelium. Albrecht
von Graefes Arch Klin Exp Ophthalmol 1981; 215: 233-242
Reference Ris Wihthout Link
- 26
Maser MD,
Trimble JJ.
Rapid chemical dehydration of biologic samples for scanning electron microscopy using
2,2-dimethoxypropane. J Histochem Cytochem 1977; 25: 247-251
Reference Ris Wihthout Link
- 27
Conway K,
Kiernan JA.
Chemical dehydration of specimens with 2,2-dimethoxypropane (DMP) for paraffin processing
of animal tissues: Practical and economic advantages over dehydration in ethanol.
Biotech Histochem 1999; 74: 20-26
Reference Ris Wihthout Link
- 28
Sun N,
Shibata B,
Hess JF.
et al.
An alternative means of retaining ocular structure and improving immunoreactivity
for light microscopy studies. Mol Vis 2015; 21: 428-442
Reference Ris Wihthout Link
- 29
Tran H,
Jan NJ,
Hu D.
et al.
Formalin Fixation and Cryosectioning Cause only Minimal Changes in Shape or Size of
Ocular Tissues. Sci Rep 2017; 7: 1-11
Reference Ris Wihthout Link
- 30
Hedrick BP,
Yohe L,
Vander Linden A.
et al.
Assessing Soft-Tissue Shrinkage Estimates in Museum Specimens Imaged With Diffusible
Iodine-Based Contrast-Enhanced Computed Tomography (diceCT). Microsc Microanal 2018;
24: 284-291
Reference Ris Wihthout Link
- 31
Dawood Y,
Hagoort J,
Siadari BA.
et al.
Reducing soft-tissue shrinkage artefacts caused by staining with Lugolʼs solution.
Sci Rep 2021; 11: 1-13
Reference Ris Wihthout Link
- 32
Wree A,
Schulze M,
Keiler J.
Macroscopic anatomy of the nasolacrimal system. In: Anatomische Gesellschaft: 113th
Annual Meeting, Rostock 2018. Lecture Abstracts 2018
Reference Ris Wihthout Link
- 33
Bissinger O,
Götz C,
Wolff KD.
et al.
Fully automated segmentation of callus by micro-CT compared to biomechanics. J Orthop
Surg Res 2017; 12: 1-9
Reference Ris Wihthout Link
- 34
Ferl GZ,
Barck KH,
Patil J.
et al.
Automated segmentation of lungs and lung tumors in mouse micro-CT scans. iScience
2022; 25: 105712
Reference Ris Wihthout Link
- 35
Vincenzi E,
Fantazzini A,
Basso C.
et al.
A fully automated deep learning pipeline for micro-CT-imaging-based densitometry of
lung fibrosis murine models. Respir Res 2022; 23: 1-14
Reference Ris Wihthout Link
- 36
Malimban J,
Lathouwers D,
Qian H.
et al.
Deep learning-based segmentation of the thorax in mouse micro-CT scans. Sci Rep 2022;
12: 1-12
Reference Ris Wihthout Link
- 37
Barillot C,
Gibaud B,
Lis O.
et al.
Computer graphics in medicine: a survey. Crit Rev Biomed Eng 1988; 15: 269-307
Reference Ris Wihthout Link
- 38
Palladino A,
Salerno A,
Crasto A.
et al.
Integration of micro-CT and histology data for vasculature morpho-functional analysis
in tissue regeneration. Ann Anat 2023; 245: 152019
Reference Ris Wihthout Link
- 39
Chicherova N,
Hieber SE,
Khimchenko A.
et al.
Automatic deformable registration of histological slides to µCT volume data. J Microsc
2018; 271: 49-61
Reference Ris Wihthout Link
- 40
Lupariello F,
Genova T,
Mussano F.
et al.
Micro-CT processingʼs effects on microscopic appearance of human fetal cardiac samples.
Leg Med 2021; 53: 101934
Reference Ris Wihthout Link