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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
both unscattered electrons (brightfield model) and scattered electrons (darkfield model) can be collected. However, resolution in the
FESEM is limited only by beam size. The absence of a lens beneath the sample in the FE-SEM eliminates spherical aberrations
induced by the scattering angle and chromic aberrations due to energy loss. In the FE-SEM the maximum beam energy is limited
typically to 30 kV and there is no need for a CCD or a film camera.
The maximum beam energy of the FE-SEM limits the maximum beam penetration depth which depends on material composition
mainly. Heavy metal stained materials like biological samples are ideal candidates for low voltage STEM in the FE-SEM.
Fig. 1. The unique arrangement of both brightfield (BF) and darkfield (DF) diodes in the ZEISS Multi-Mode STEM detector. BF and
DF electrons can be collected simultaneously and processed together. BF inverted DF is a typical configuration for large fields of
view in excess of 100 microns with even illumination.
Resolution in STEM mode can be as high as 0.6 nm with FE-SEM approaching that of the TEM and STEM image quality may
actually exceed that of TEM in some aspects (e.g. contrast). A few examples show image quality easily attainable with the FE-SEM
in STEM mode in the following.
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
Fig. 2. The STEM image of 10 nm immunogold labeled rat hypothalamus in Lowicryl HM20 epoxy, no post stain.
Fig. 3. The STEM image of a myelinated axon sheath in unstained rat hippocampus ultra section with 3 nm spacing.
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
Fig. 4. The images of a multi sample carousel holder, a TEM grid carousel holder and a formvar slot grid with17 serial ultra sections.
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
allows definition of the mosaic parameters and auto functions. As shown in Figure 5, the mosaic dimension, single tile pixel size,
single tile resolution, dwell time and overlapping area size etc., can be chosen in the initial set-up procedure according to the
requirements of the job to be performed. In general, the application procedure of the ATLAS is as follows:
Load the sample into the FE-SEM. Choose Create a mosaic from the ATLAS user interface followed by the set-up of
Mosaic Batch Processing including Stage Location, Mosaic Parameters and Auto Functions etc.
After defining the mosaic job parameters according to the requirements, the image acquisition will automatically start by
simply clicking Execute.
The generated image tiles with high pixel resolution can be handled and stitched by the integrated viewer software.
The stitched mosaic can be viewed and navigated, output and saved by the viewer with the required resolution.
Fig. 5. The mosaic option panel shows the arbitrary parameter setting and auto functions.
Figure 6 shows a typical single site 6 2 mosaic covering one 250 micron wide ultra section. Each of the 12 tiles has a 48-micron
field of view, and a single 24 k 24 k pixel image displays 2 nm pixel resolution.
Fig. 6. The 6 2 mosaic image of ultramicrotome cross section of rat hippocampus recorded with STEM detector.
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
Fig. 8. The configuration of the multi carousel holder with 12 grids inside the stage navigation of Smart SEM.
Software
Once the automated job is finished, the generated image tiles can be viewed and stitched together by the integrated Viewer software
to obtain a panoramic image of a large area. The Viewer allows the user to efficiently open, stitch, navigate, output/save and
intelligently re-render the large two dimensional datasets produced by ATLAS.
Summary
FE-SEM based STEM imaging in combination with ATLAS is a new high resolution, high throughput imaging technique for tissue
samples and provides an alternative to traditional TEM imaging of biological samples. You are no longer limited to only imaging a
small area of the sample in high resolution. Here FE-SEM-based STEM in combination with ATLAS is a superior solution to large
volume image acquisition.
Acknowledgments
We appreciate Dr. Doug Wei and his team for their pioneering ideas and full support in the ATLAS development.
John Mendenhall, Center for Learning and Memory, University of Texas at Austin is gratefully acknowledged for providing the
samples and significant discussion.
Source: "Automated Extreme Field of View Low Voltage Multi-Mode STEM Imaging of Biological Ultramicrotome Cross Sections with ATLAS" by
Carl Zeiss
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
Date Added: Nov 16, 2010 | Updated: Jun 11, 2013
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High Resolution Imaging of Biological Samples Using Scanning Electron Microscopes and The ATLAS by Carl Zeiss
Carl-Zeiss-Promenade 10
Jena
07745
Germany
PH: +49 (1803) 33 63 34
Email: micrscopy@zeiss.com
Visit Carl Zeiss Microscopy GmbH Website
Primary Activity
Microscope solutions for life science and materials research
Company Background
The Microscopy business group at Carl Zeiss is the world's only manufacturer of both light and electron microscopes. The company's
extensive portfolio enables research and routine applications in the life and materials sciences.
Their product range includes light and laser scanning microscopes, electron and ion microscopes and spectrometer modules. Users
are supported for software for system control, image capture and editing.
Carl Zeiss Microscopy has sales companies in 33 countries. Application and service specialists support customers around the globe
in demo centres and on site.
The business group is headquartered in Jena. Additional production and development sites can be found in Oberkochen, Gttingen
and Munich, as well as in the UK and the USA. The company has around 2,500 employees and generates revenue of 620 million
euros.
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