title: A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario.
abstract: BACKGROUND
  In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples.
  
  
  RESULTS
  We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system.
  
  
  CONCLUSIONS
  The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations.
authors.affiliation: VetImaging, VetCore Facility for Research, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. stephan.handschuh@vetmeduni.ac.at.
authors.forename: Stephan
authors.initials: S
authors.lastname: Handschuh
authors.forename: Natalie
authors.initials: N
authors.lastname: Baeumler
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwaha
authors.forename: Bernhard
authors.initials: B
authors.lastname: Ruthensteiner
references.citation: IEEE Trans Med Imaging. 1997 Apr;16(2):187-98
references.pmid: 9101328
references.citation: Nat Methods. 2012 Jun 10;9(7):717-20
references.pmid: 22688414
references.citation: Stain Technol. 1976 Sep;51(5):275-6
references.pmid: 793092
references.citation: J Neurosci Methods. 2008 Jun 15;171(1):93-7
references.pmid: 18400304
references.citation: PLoS One. 2010 Feb 03;5(2):e9014
references.pmid: 20140253
references.citation: J Histochem Cytochem. 1993 May;41(5):769-72
references.pmid: 8468459
references.citation: Cold Spring Harb Protoc. 2011 Dec 01;2011(12):1462-71
references.pmid: 22135670
references.citation: BMC Physiol. 2009 Jun 22;9:11
references.pmid: 19545439
references.citation: J Microsc. 2007 Jul;227(Pt 1):51-71
references.pmid: 17635659
references.citation: BMC Dev Biol. 2010 Apr 21;10:41
references.pmid: 20409315
references.citation: PLoS One. 2009 Nov 06;4(11):e7716
references.pmid: 19893745
references.citation: Front Zool. 2012 Sep 14;9(1):23
references.pmid: 22973977
references.citation: Front Neuroanat. 2010 Jun 14;4:24
references.pmid: 20676237
references.citation: J Neurosci Methods. 2008 Jun 30;171(2):207-13
references.pmid: 18462802
references.citation: J Cell Biol. 1974 Apr;61(1):261-4
references.pmid: 4594615
references.citation: Nat Protoc. 2012 Jan 12;7(2):193-206
references.pmid: 22240582
references.citation: Methods Cell Biol. 2010;96:331-61
references.pmid: 20869530
references.citation: Neuron. 2007 Jul 5;55(1):25-36
references.pmid: 17610815
references.citation: J Microsc. 2009 Sep;235(3):239-40
references.pmid: 19754718
references.citation: PLoS Genet. 2006 Apr;2(4):e61
references.pmid: 16683035
references.citation: Methods Cell Biol. 2012;111:325-56
references.pmid: 22857936
references.citation: Curr Opin Struct Biol. 2011 Oct;21(5):686-93
references.pmid: 21782417
references.citation: J Vis Exp. 2011 Jul 06;(53):e2588
references.pmid: 21775953
references.citation: J Cell Biochem Suppl. 2002;39:116-24
references.pmid: 12552611
references.citation: Stain Technol. 1972 May;47(3):115-8
references.pmid: 5019001
references.citation: J R Microsc Soc. 1967;87(3):485-7
references.pmid: 5582820
references.citation: Annu Rev Biomed Eng. 2011 Aug 15;13:531-52
references.pmid: 21756145
references.citation: J Histochem Cytochem. 2013 Apr;61(4):263-71
references.pmid: 23264636
references.citation: J Microsc. 2009 Sep;235(3):308-21
references.pmid: 19754725
references.citation: J Morphol. 2013 Apr;274(4):456-66
references.pmid: 23280634
references.citation: Virchows Arch. 2011 Jul;459(1):99-108
references.pmid: 21660521
references.citation: J Morphol. 2011 Aug;272(8):972-86
references.pmid: 21598290
references.citation: Stain Technol. 1965 Sep;40(5):259-63
references.pmid: 5326579
references.citation: Med Image Anal. 1996 Mar;1(1):35-51
references.pmid: 9873920
references.citation: PLoS Biol. 2010 Oct 05;8(10):
references.pmid: 20957184
references.citation: Dev Dyn. 2009 Mar;238(3):632-40
references.pmid: 19235724
references.citation: Bioinformatics. 2010 Jun 15;26(12):i57-63
references.pmid: 20529937
references.citation: Circ Cardiovasc Imaging. 2010 May;3(3):314-22
references.pmid: 20190279