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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

TEM section 3D registration. A. Template from an LM section that was used for TEM re-sectioning. B. TEM image co-registered to the LM template in Photoshop. TEM image layer set to 50% opacity. C. Registered TEM image with (nearly) black background as re-imported into AMIRA®. D. Voxel dimensions (0.413×0.413 μm in X and Y axes) of the cropped LM template before resampling to TEM resolution. E. Voxel dimensions (0.025×0.025 μm) of the cropped LM template after resampling to TEM resolution (same as in 3D registered TEM image).
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Figure 5: TEM section 3D registration. A. Template from an LM section that was used for TEM re-sectioning. B. TEM image co-registered to the LM template in Photoshop. TEM image layer set to 50% opacity. C. Registered TEM image with (nearly) black background as re-imported into AMIRA®. D. Voxel dimensions (0.413×0.413 μm in X and Y axes) of the cropped LM template before resampling to TEM resolution. E. Voxel dimensions (0.025×0.025 μm) of the cropped LM template after resampling to TEM resolution (same as in 3D registered TEM image).

Mentions: Prior to the registration of a TEM image into the 3D network, we created templates from the LM sections that were actually used for TEM re-sectioning (Figure 4F). To achieve this, the selected LM section image was isolated from the already registered LM stack with the Crop Editor and saved as separate AM file in AMIRA®. For each TEM image to be registered, the LM section was cropped to the specific region of interest (Figure 5A) again with the Crop Editor. Subsequently the resolution of the dataset (crop of a single slice) was strongly increased (Compute, Resample) to reach the voxel (pixel) size of the respective TEM image (calculated from TEM scale bar) (Figure 5D and 5E). After resampling of the template, the transformation details had to be restored from the original cropped image using the copy/paste function of the Transform Editor, and the resampled and transformed template was saved in AM format. The template was then exported in 2D TIFF format and loaded into Photoshop CS5. Thereafter the TEM image was loaded into the same Photoshop document into a new layer and aligned to the LM template using the Auto-Align-Layers function. To check the quality of alignment, the TEM image layer was inspected with 50% opacity (Figure 5B). Distortion of TEM images relative to the LM images was mostly negligible and no elastic registration appeared necessary. In a few cases the size of TEM images had to be slightly rescaled (isotropically) to match the LM image exactly. A black background layer was inserted below the TEM image layer, and the 8-bit histogram of the TEM image was modified to 10–255 using a Levels layer to provide that all areas of the TEM image remain visible at visualization in AMIRA® (see below). Eventually the registered TEM image was combined with the fully black background and saved as TIFF file (Figure 5C; note that it is crucial that the canvas size in the Photoshop document remains unchanged during this procedure). This image was then re-imported into AMIRA®. The position coordinates within the scenery were restored from the resampled template with the Crop Editor, and transformation coordinates were reused from the resampled template again using the copy/paste function of the Transform Editor. Finally the TEM image was saved in Amira mesh (AM) format.


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)

TEM section 3D registration. A. Template from an LM section that was used for TEM re-sectioning. B. TEM image co-registered to the LM template in Photoshop. TEM image layer set to 50% opacity. C. Registered TEM image with (nearly) black background as re-imported into AMIRA®. D. Voxel dimensions (0.413×0.413 μm in X and Y axes) of the cropped LM template before resampling to TEM resolution. E. Voxel dimensions (0.025×0.025 μm) of the cropped LM template after resampling to TEM resolution (same as in 3D registered TEM image).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: TEM section 3D registration. A. Template from an LM section that was used for TEM re-sectioning. B. TEM image co-registered to the LM template in Photoshop. TEM image layer set to 50% opacity. C. Registered TEM image with (nearly) black background as re-imported into AMIRA®. D. Voxel dimensions (0.413×0.413 μm in X and Y axes) of the cropped LM template before resampling to TEM resolution. E. Voxel dimensions (0.025×0.025 μm) of the cropped LM template after resampling to TEM resolution (same as in 3D registered TEM image).
Mentions: Prior to the registration of a TEM image into the 3D network, we created templates from the LM sections that were actually used for TEM re-sectioning (Figure 4F). To achieve this, the selected LM section image was isolated from the already registered LM stack with the Crop Editor and saved as separate AM file in AMIRA®. For each TEM image to be registered, the LM section was cropped to the specific region of interest (Figure 5A) again with the Crop Editor. Subsequently the resolution of the dataset (crop of a single slice) was strongly increased (Compute, Resample) to reach the voxel (pixel) size of the respective TEM image (calculated from TEM scale bar) (Figure 5D and 5E). After resampling of the template, the transformation details had to be restored from the original cropped image using the copy/paste function of the Transform Editor, and the resampled and transformed template was saved in AM format. The template was then exported in 2D TIFF format and loaded into Photoshop CS5. Thereafter the TEM image was loaded into the same Photoshop document into a new layer and aligned to the LM template using the Auto-Align-Layers function. To check the quality of alignment, the TEM image layer was inspected with 50% opacity (Figure 5B). Distortion of TEM images relative to the LM images was mostly negligible and no elastic registration appeared necessary. In a few cases the size of TEM images had to be slightly rescaled (isotropically) to match the LM image exactly. A black background layer was inserted below the TEM image layer, and the 8-bit histogram of the TEM image was modified to 10–255 using a Levels layer to provide that all areas of the TEM image remain visible at visualization in AMIRA® (see below). Eventually the registered TEM image was combined with the fully black background and saved as TIFF file (Figure 5C; note that it is crucial that the canvas size in the Photoshop document remains unchanged during this procedure). This image was then re-imported into AMIRA®. The position coordinates within the scenery were restored from the resampled template with the Crop Editor, and transformation coordinates were reused from the resampled template again using the copy/paste function of the Transform Editor. Finally the TEM image was saved in Amira mesh (AM) format.

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