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A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario.

Handschuh S, Baeumler N, Schwaha T, Ruthensteiner B - Front. Zool. (2013)

Bottom Line: Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: VetImaging, VetCore Facility for Research, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. stephan.handschuh@vetmeduni.ac.at.

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.

No MeSH data available.


Related in: MedlinePlus

MicroCT data (volume rendering) and LM data (surface rendering and orthoslices) combined. AMIRA® software visualization. A. View from the left side, microCT data (volren module). B. MicroCT data with high transparency and transparent orthoslices of both LM sections used for TEM re-sectioning with TEM sections. C, D. Right half of microCT data, surfaces of various organs and orthoslices of LM sections used for TEM re-sectioning. C. View from left side. D. View from posterior. F–G. Same as C, D but without orthoslices. E. View from anterior. F. View from obliquely left. G. View from posterior. cg, cerebral ganglion; dg, digestive gland; f, foot; g, gill; gv, gill vessels; i, intestine; me, mantle edge; mu, muscle tissue; oe, oesphagus; ol, oral lappets; pe, pericardium; po, periostracum and periostracal structures; re, rectum; vn, visceral nerve.
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Figure 7: MicroCT data (volume rendering) and LM data (surface rendering and orthoslices) combined. AMIRA® software visualization. A. View from the left side, microCT data (volren module). B. MicroCT data with high transparency and transparent orthoslices of both LM sections used for TEM re-sectioning with TEM sections. C, D. Right half of microCT data, surfaces of various organs and orthoslices of LM sections used for TEM re-sectioning. C. View from left side. D. View from posterior. F–G. Same as C, D but without orthoslices. E. View from anterior. F. View from obliquely left. G. View from posterior. cg, cerebral ganglion; dg, digestive gland; f, foot; g, gill; gv, gill vessels; i, intestine; me, mantle edge; mu, muscle tissue; oe, oesphagus; ol, oral lappets; pe, pericardium; po, periostracum and periostracal structures; re, rectum; vn, visceral nerve.

Mentions: MicroCT, LM, and TEM data were displayed simultaneously in a single AMIRA® 3D scene (Network) (Figures 6, 7, 8) using a combination of different standard visualization devices for viewing volume data, polygonal surfaces, and individual slices. For volume data from microCT and LM sections both the Voltex (volume rendering via texture mapping) and the Volren module (volume rendering via ray casting) was used. Surface mesh files were rendered with the SurfaceView tool in Direct Normals mode. For visualizing LM and TEM sections OrthoSlices were used. In the case of TEM sections, the OrthoSlice was combined with a specifically adapted grayscale colormap with standard gray values and a transparency function where opacity for input gray values 0–9 is set to 0, and opacity in gray values 10–255 is set to 255. This yielded complete transparency in the surrounding area (black background, Figure 5C) and total visibility of the aligned TEM images.


A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario.

Handschuh S, Baeumler N, Schwaha T, Ruthensteiner B - Front. Zool. (2013)

MicroCT data (volume rendering) and LM data (surface rendering and orthoslices) combined. AMIRA® software visualization. A. View from the left side, microCT data (volren module). B. MicroCT data with high transparency and transparent orthoslices of both LM sections used for TEM re-sectioning with TEM sections. C, D. Right half of microCT data, surfaces of various organs and orthoslices of LM sections used for TEM re-sectioning. C. View from left side. D. View from posterior. F–G. Same as C, D but without orthoslices. E. View from anterior. F. View from obliquely left. G. View from posterior. cg, cerebral ganglion; dg, digestive gland; f, foot; g, gill; gv, gill vessels; i, intestine; me, mantle edge; mu, muscle tissue; oe, oesphagus; ol, oral lappets; pe, pericardium; po, periostracum and periostracal structures; re, rectum; vn, visceral nerve.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750762&req=5

Figure 7: MicroCT data (volume rendering) and LM data (surface rendering and orthoslices) combined. AMIRA® software visualization. A. View from the left side, microCT data (volren module). B. MicroCT data with high transparency and transparent orthoslices of both LM sections used for TEM re-sectioning with TEM sections. C, D. Right half of microCT data, surfaces of various organs and orthoslices of LM sections used for TEM re-sectioning. C. View from left side. D. View from posterior. F–G. Same as C, D but without orthoslices. E. View from anterior. F. View from obliquely left. G. View from posterior. cg, cerebral ganglion; dg, digestive gland; f, foot; g, gill; gv, gill vessels; i, intestine; me, mantle edge; mu, muscle tissue; oe, oesphagus; ol, oral lappets; pe, pericardium; po, periostracum and periostracal structures; re, rectum; vn, visceral nerve.
Mentions: MicroCT, LM, and TEM data were displayed simultaneously in a single AMIRA® 3D scene (Network) (Figures 6, 7, 8) using a combination of different standard visualization devices for viewing volume data, polygonal surfaces, and individual slices. For volume data from microCT and LM sections both the Voltex (volume rendering via texture mapping) and the Volren module (volume rendering via ray casting) was used. Surface mesh files were rendered with the SurfaceView tool in Direct Normals mode. For visualizing LM and TEM sections OrthoSlices were used. In the case of TEM sections, the OrthoSlice was combined with a specifically adapted grayscale colormap with standard gray values and a transparency function where opacity for input gray values 0–9 is set to 0, and opacity in gray values 10–255 is set to 255. This yielded complete transparency in the surrounding area (black background, Figure 5C) and total visibility of the aligned TEM images.

Bottom Line: Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: VetImaging, VetCore Facility for Research, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. stephan.handschuh@vetmeduni.ac.at.

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.

No MeSH data available.


Related in: MedlinePlus