Need for Large Images
XFOV - STEM in SEM
Advantages of SEM-VE
The SEM Visualization Engine (SEM-VE) combines a 16 bit scan
generator and dual super-sampling signal acquisition hardware with
image processing and control software for your Zeiss electron
microscope. The SEM-VE system enables acquisition of images up to 32k x
32k pixels (one gigapixel image size), with dwell times from 100 ns to
> 100 s, adjustable in 100 ns increments. Images may be saved with
eight or sixteen bits of intensity. The SEM-VE "Mosaic Tool" is
designed to create large image montages and is highly integrated with
The Mosaic Tool enables the automated acquisition of multi-image
Mosaics at multiple sites on the sample, automatically moving from
"image Tile" to Tile, and Mosaic site to site, executing SmartSEM
autofunctions such as Focus, Stigmation, Brightness, etc. as required.
The end result is an "Extreme Field of View" image montage that can
cover optical microscope scale (or even naked eye scale) areas of your
sample, at SEM nanometer scale resolution.
Given suitable samples, unattended operation can be performed over a
period of days, automatically acquiring Terabytes of image data at
rates up to 30 Gigabytes of image data per hour. This allows the user
to collect data from a given region at a resolution approximately seven
orders of magnitude smaller than the region. For example it is possible
to acquire a 40 square millimeter region, at 4 nanometer resolution, in
under 4 days.
Single 30 nm thick section of mouse brain imaged with
backscattered electrons at 8 kV using a Zeiss Sigma FE-SEM. Original
image resolution was 4 nm per pixel. Red arrows point to areas where
one neuron contacts and transfers information to another.
Extreme field of view image montages improve the complete
understanding of structures by providing detailed image information
from the macro scale to the nanoscale.
Need for Large Images
Why acquire "EXTREMELY LARGE" images?
- Reduces the number of tiles to acquire, reducing stage motion
delay and more importantly reducing the areal fraction of each image
"lost" to overlap. For a 4 nm pixel size and a 2 µm stage accuracy,
overlap can be 66% of the image area at 3k x 2k (33% new data), but
only 6% of the image area at 32k x 32k (94% new data / image) thus a
2.5 mm x 2.5 mm mosaic at 4 nm resolution would require ~92,000 images
with a conventional frame store, but only 400 images when using the
- Reduces the number of overlap "seams", leading to less beam
damage and degradation of the sample.
- These factors greatly reduce computational complexity -
considerably easier to stitch and align when working with 400 images
rather than 92,000. This reduction in computational complexity becomes
even more critical when three dimensional alignment of serial sections
Image tile boundaries are hidden.
The position of image tile boundaries are displayed
A 65 nm technology node graphics processor integrated circuit,
stripped to its silicon substrate with HF acid etching.
The mosaic consists of 49 images, each ~500 Megapixels,
automatically stitched by the VE-Viewer into a ~1/3 mm x 1/3 mm mosaic.
This mosaic was acquired stepping at an angle, with scan rotation used
to align image features to the scan axes, in order to accentuate the
stitching for illustrative purposes.
A magnified view of the junction between four tiles
as shown above, with tile boundaries displayed in white, as otherwise
the boundaries between tiles are virtually seamless.
A sample of the high resolution detail visible across
the entire Extreme Field of View mosaic.
XFOV - STEM in SEM
By using the GEMINI® Multi-mode STEM detection system, the
information limit for the SUPRA™
FE-SEM can be extended beyond the nanometer range. A resolution of
0.8 nm is now readily attainable and gives additional nano scale
information. The quality of the image obtained with the STEM unit are
similar to images obtained by a TEM with a scanning attachment.
STEM-in-SEM images acquired on a Zeiss SUPRA™
40 equipped with SEM-VE. Images show a single 45 nm thick
post-section stained epoxy ultra section of perfused rat hippocampus. A
series of images were automatically acquired from multiple sites on
multiple TEM grids. (A) Low magnification overview image of a single
mosaic site consisting of 6 x 2 tiles, each 49 µm x 49 µm, acquired
using the GEMINI® STEM detection system in a SUPRA™
FE-SEM. Each image tile was acquired at 2 nm per pixel. (B) A
single 49 µm SEM-in-STEM image, acquired at 2 nm per pixel. The inset
square illustrates the comparative size of a TEM image acquired on a 4k
x 4k camera, also at 2 nm per pixel. Acquiring larger fields of view
reduces both distortion and the requirements for image stitching. (C)
Detail from an approximately 9 µm x 9 µm region of Image B.
Note the image quality is comparable to results obtained by
conventional TEM. At full resolution, key organelles are readily
resolved including (D) post synaptic densities at synapses, (E)
polyribosomes and (F) cross-sectioned microtubules suitable for serial
tracing and dense reconstruction.
Advantages of SEM-VE
How Does SEM-VE Help to Solve Your Problems
- Acquire images up to 1 Gigapixel in a single scan, increased from
previous ~7 Megapixel maximum.
- Continuously selectable scan size from 1 x 1 to 32k x 32k pixels.
- Continuously definable field of view and pixel resolution.
- Scan rates selectable from 100 ns to >>100 s dwell per
pixel, in 100 ns increments.
- Save image data as 8 or 16 bit TIFF files.
- Automation enables unattended operation, with automatic stage
motion, focus, stigmation, brightness and contrast adjustment as
- Acquire and stitch high resolution image Mosaics at optical
fields of view with nanometer resolution.
- Includes the VE-Viewer, an image viewer tailored to efficiently
handle the multi-gigabyte Mosaics produced by SEM-VE. The VE-Viewer
allows the user to open, stitch, navigate and intelligently re-render
the large 2D datasets produced by SEM-VE.
A high magnification excerpt from the long image.
A long, narrow single image approximately 1/3 mm x 20 µm in size
acquired at 10 nm per pixel resolution and 200 ns dwell time. This
image took approximately 15 seconds to acquire.
SEM-VE is available on new and currently installed instruments as a
Source: "ZEISS XFOV Zeiss eXtreme Field of View" by
For more information on this source, please visit Carl Zeiss.