Limits...
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.


Re-sectioning of LM sections for TEM. A. Excerpt of the LM section series on slide. B. Drop of distilled water is placed atop the section to be lifted. C. Section detached. D. Section is picked up with the tip of a needle. E. Block that has been perfectly smoothened by sectioning. F. Drop of distilled water placed atop the block. G. Section placed in drop. H. Section dried on block surface. I. Block trimmed for TEM re-sectioning.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750762&req=5

Figure 3: Re-sectioning of LM sections for TEM. A. Excerpt of the LM section series on slide. B. Drop of distilled water is placed atop the section to be lifted. C. Section detached. D. Section is picked up with the tip of a needle. E. Block that has been perfectly smoothened by sectioning. F. Drop of distilled water placed atop the block. G. Section placed in drop. H. Section dried on block surface. I. Block trimmed for TEM re-sectioning.

Mentions: The specimen was initially sectioned for LM (“semithin”) at a thickness of 1.5 μm using a Histo Jumbo diamond knife (Diatome AG, Biel, Switzerland) and with ribbon formation of sections (see [25] for protocol). The section ribbons were applied to conventional (un-pretreated) microscope slides that were cleaned as described by [25]. Since some sections later had to be detached for TEM investigation, the series was left uncovered with no mounting medium and no coverslip applied. Re-sectioning of LM sections for TEM (Figure 3) followed the method described by Campbell & Hermans [26]. An empty resin block was trimmed so that it had a cutting surface as large as the LM section to be re-mounted. The empty block was sectioned on an ultramicrotome with a diamond knife to obtain a smooth surface (Figure 3E). The holder with the block was then removed from the microtome and placed with the cutting surface facing up and a drop of distilled water was placed on it (Figure 3F). Subsequently the LM sections selected for re-sectioning were removed from the microscope slide by placing a drop of distilled water at the edge of the section (Figure 3B). The section then was removed from the slide by gently detaching it from the side with the tip of a fine needle and simultaneously dragging the water underneath the section (Figure 3C). This resulted in the section floating on the surface of the drop. From here sections were picked up with tip of a needle (Figure 3D) and transferred to the drop on top of the cutting surface of the block (Figure 3G). Excess water was removed with the help of filter paper to prevent wrinkles in the sections. Thereafter the block was placed in an oven at 40°C for at least 30 minutes to increase adhesion of the section to the block (Figure 3H). Prior to TEM sectioning the block was trimmed until the cutting face was smaller than the LM section before re-mounting (Figure 3I); every edge of the LM section was cropped.


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)

Re-sectioning of LM sections for TEM. A. Excerpt of the LM section series on slide. B. Drop of distilled water is placed atop the section to be lifted. C. Section detached. D. Section is picked up with the tip of a needle. E. Block that has been perfectly smoothened by sectioning. F. Drop of distilled water placed atop the block. G. Section placed in drop. H. Section dried on block surface. I. Block trimmed for TEM re-sectioning.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Re-sectioning of LM sections for TEM. A. Excerpt of the LM section series on slide. B. Drop of distilled water is placed atop the section to be lifted. C. Section detached. D. Section is picked up with the tip of a needle. E. Block that has been perfectly smoothened by sectioning. F. Drop of distilled water placed atop the block. G. Section placed in drop. H. Section dried on block surface. I. Block trimmed for TEM re-sectioning.
Mentions: The specimen was initially sectioned for LM (“semithin”) at a thickness of 1.5 μm using a Histo Jumbo diamond knife (Diatome AG, Biel, Switzerland) and with ribbon formation of sections (see [25] for protocol). The section ribbons were applied to conventional (un-pretreated) microscope slides that were cleaned as described by [25]. Since some sections later had to be detached for TEM investigation, the series was left uncovered with no mounting medium and no coverslip applied. Re-sectioning of LM sections for TEM (Figure 3) followed the method described by Campbell & Hermans [26]. An empty resin block was trimmed so that it had a cutting surface as large as the LM section to be re-mounted. The empty block was sectioned on an ultramicrotome with a diamond knife to obtain a smooth surface (Figure 3E). The holder with the block was then removed from the microtome and placed with the cutting surface facing up and a drop of distilled water was placed on it (Figure 3F). Subsequently the LM sections selected for re-sectioning were removed from the microscope slide by placing a drop of distilled water at the edge of the section (Figure 3B). The section then was removed from the slide by gently detaching it from the side with the tip of a fine needle and simultaneously dragging the water underneath the section (Figure 3C). This resulted in the section floating on the surface of the drop. From here sections were picked up with tip of a needle (Figure 3D) and transferred to the drop on top of the cutting surface of the block (Figure 3G). Excess water was removed with the help of filter paper to prevent wrinkles in the sections. Thereafter the block was placed in an oven at 40°C for at least 30 minutes to increase adhesion of the section to the block (Figure 3H). Prior to TEM sectioning the block was trimmed until the cutting face was smaller than the LM section before re-mounting (Figure 3I); every edge of the LM section was cropped.

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.