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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.


General workflow for combining microCT, LM and TEM. Red arrows, volume data from microCT; blue arrows, LM section data; green arrows, TEM section data; green encircled, material and (interim) results; orange encircled, microscopic equipment; black encircled, software systems. A. EM fixed specimen embedded in epoxy resin block, anterior one used in study. B. Block trimmed and anterior portion detached, after microCT scan. C. MicroCT. D. Volume rendering with Drishti. E. Volume rendering with AMIRA®. F. Microtome for LM sectioning. G. LM section series. H. Light microscope. I. Surface rendering with AMIRA®. J. Remounting of LM sections for ultrathin sectioning. K. Ultramicrotome. L. TEM. M. 2D alignment for figure plates. N. 3D registration with AMIRA®. O. Combined 3D visualization of all datasets.
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Figure 1: General workflow for combining microCT, LM and TEM. Red arrows, volume data from microCT; blue arrows, LM section data; green arrows, TEM section data; green encircled, material and (interim) results; orange encircled, microscopic equipment; black encircled, software systems. A. EM fixed specimen embedded in epoxy resin block, anterior one used in study. B. Block trimmed and anterior portion detached, after microCT scan. C. MicroCT. D. Volume rendering with Drishti. E. Volume rendering with AMIRA®. F. Microtome for LM sectioning. G. LM section series. H. Light microscope. I. Surface rendering with AMIRA®. J. Remounting of LM sections for ultrathin sectioning. K. Ultramicrotome. L. TEM. M. 2D alignment for figure plates. N. 3D registration with AMIRA®. O. Combined 3D visualization of all datasets.

Mentions: The specimen was fixed and embedded using standard procedures for transmission electron microscopy (TEM) investigation. Subsequently it was scanned by microCT. This was followed by sectioning for light microscopical (LM) investigation. Based on serial LM images and image segmentation, a 3D surface model of organ systems was generated. This permitted precisely tracking down ROIs and thus individual LM sections for fine structural investigation. Selected sections were re-sectioned for TEM. The datasets gained from microCT (volume dataset), LM (volume dataset, surface model) and TEM (individual sections) were co-registered and combined to a single visualization using AMIRA® (version 5.3.3, Visage Imaging, Berlin, Germany) (Figure 1).


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)

General workflow for combining microCT, LM and TEM. Red arrows, volume data from microCT; blue arrows, LM section data; green arrows, TEM section data; green encircled, material and (interim) results; orange encircled, microscopic equipment; black encircled, software systems. A. EM fixed specimen embedded in epoxy resin block, anterior one used in study. B. Block trimmed and anterior portion detached, after microCT scan. C. MicroCT. D. Volume rendering with Drishti. E. Volume rendering with AMIRA®. F. Microtome for LM sectioning. G. LM section series. H. Light microscope. I. Surface rendering with AMIRA®. J. Remounting of LM sections for ultrathin sectioning. K. Ultramicrotome. L. TEM. M. 2D alignment for figure plates. N. 3D registration with AMIRA®. O. Combined 3D visualization of all datasets.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: General workflow for combining microCT, LM and TEM. Red arrows, volume data from microCT; blue arrows, LM section data; green arrows, TEM section data; green encircled, material and (interim) results; orange encircled, microscopic equipment; black encircled, software systems. A. EM fixed specimen embedded in epoxy resin block, anterior one used in study. B. Block trimmed and anterior portion detached, after microCT scan. C. MicroCT. D. Volume rendering with Drishti. E. Volume rendering with AMIRA®. F. Microtome for LM sectioning. G. LM section series. H. Light microscope. I. Surface rendering with AMIRA®. J. Remounting of LM sections for ultrathin sectioning. K. Ultramicrotome. L. TEM. M. 2D alignment for figure plates. N. 3D registration with AMIRA®. O. Combined 3D visualization of all datasets.
Mentions: The specimen was fixed and embedded using standard procedures for transmission electron microscopy (TEM) investigation. Subsequently it was scanned by microCT. This was followed by sectioning for light microscopical (LM) investigation. Based on serial LM images and image segmentation, a 3D surface model of organ systems was generated. This permitted precisely tracking down ROIs and thus individual LM sections for fine structural investigation. Selected sections were re-sectioned for TEM. The datasets gained from microCT (volume dataset), LM (volume dataset, surface model) and TEM (individual sections) were co-registered and combined to a single visualization using AMIRA® (version 5.3.3, Visage Imaging, Berlin, Germany) (Figure 1).

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.