3View®2 - Electron Microscopy Core Facility – University of Missouri

3View 2
Features and Capabilities
Specifications, Recommendations and Ordering
®
Specification/
Recommendation
Simplifying EM
The 3View®2 setup and operation is intuitive and simple. Removing the most difficult
step, cutting and collecting ultrathin sections, 3View®2 can turn an embedded sample into
thousands on images overnight. In addition 3View®2 can also cut thinner than a conventional
ultramictotome, cutting a sample from <15 nm to 200 nanometers with a total traverse of
600 microns.
Cutting Thin
3View®2XP has the ability to cut biological and material samples thinner than 15 nm. This is made possible by precise z
stage control, stabilization of the microtome, and a custom diamond knife. The 3View®2XP z movement is controlled by a
piezoelectric device, allowing precise movements at the nanometer scale. Adjusting the cutting thickness is controlled by
DigitalMicrograph®. In addition, the 3View®2XP stage has been stabilized to minimize vibrations reducing cutting discrepancy
thickness. Image: Mouse kidney prepared with the National Center for Microscopy and Imaging Research (NCMIR)
3View®2XP specimen preparation protocol embedded with Durcupan resin imaged with 7 nm pixels, and cut with 7 nm cuts
creating 7 nm isotropic voxels.
250 nm
3View®2XP
Recommended SEM
• Variable Pressure, Field Emission Gun SEM with a large chamber (Call Gatan for
specific SEM compatibility). A FEG or LaB6 is preferred due to long term beam
stability and brightness.
• Field Emission Gun SEM with a large chamber (Call Gatan for specific SEM compatibility).
A FEG is preferred due to long term beam stability & brightness, allowing cutting runs
on the order of 10 days or more.
Charge Neutralization
• Variable pressure SEM required; 5-40 Pa is typical at 2 kV.
• Variable pressure SEM is recommended but not required; 5-40 Pa is typical at 2 kV.
Accelerating Voltage
• 1 kV – 5 kV
• <1 kV – 5 kV
Cutting speed
• User defined: 0.1-1.2 mm/sec
• Recommended speed: 0.5-1 mm/sec
• User defined: 0.1-1.2 mm/sec
• Recommended speed: 0.5-1 mm/sec
Cut thickness
• Microtome can cut from 30 to 200 nm. 30 to 50 nm is typical with biological specimens.
• Microtome can cut from 15 to 200 nm. 25 to 50 nm is typical with biological specimens.
Knife Cutting Travel Distance
• 1.2 mm
• 1.2 mm
Z travel distance
• Maximum of 600 µm
• Maximum of 600 µm
3View® stage travel distance
• Traverses approximately +/-700 µm in X and Y.
• Traverses approximately +/-700 µm in X and Y.
SEM stage
• 3View®2 replaces SEM stage. High Stability Manual x-y- stage (movement sufficient to
cover 1 mm x 1 mm specimen block).
• 3View®2XP replaces SEM stage. High Vacuum Compatible automated x-y- stage
(movement sufficient to cover 1 mm x 1 mm specimen block)
Working distance
• Approximately 6 mm when used with Gatan BSED.
• Approximately 6 mm when used with Gatan BSED.
Low magnification field of view
• Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is
typically 1.2 mm x 1.2 mm.
• Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is
typically 1.2 mm x 1.2 mm.
Image acquisition
• Gatan DigiScan® uses SEM external scan control input with Firewire.
• 2-16 bit analog inputs can work simultaneously. 3View®2 acquisition is typically BSED
only. Gatan recommends its own BSE detector optimized for low kV image collection.
• Gatan DigiScan® uses SEM external scan control input with Firewire.
• 2-16 bit analog inputs can work simultaneously. 3View®2XP acquisition is typically
BSED only. Gatan recommends its own BSE detector optimized for low kV
image collection.
DigiScan® pixel density
3View®2 supports images up to 24k x 32k pixels. Actual image size is SEM dependent.
3View®2XP supports images up to 24k x 32k pixels. Actual image size is SEM dependent.
Pixel dwell time
• Microscope and sample dependant. 1-20 µs is typical.
• Microscope and sample dependant. 1-20 µs is typical.
Image scanning
NA
• Choice of single pass or configurable multiple frames.
Image calibration
• DigiScan® calibration is kV and magnification specific.
• DigiScan® calibration is kV and magnification specific.
SEM-PC communication
• Follows SEM protocol. Communication for kV, FOV, Magnification, Vacuum,
Beam Blanking.
• Follows SEM protocol. Communication for kV, FOV, Magnification, Vacuum,
Beam Blanking.
3View® setup
• Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom
microscope with chamber door at air.
• Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom
microscope with chamber door at air.
3View® operation
• Unattended, once setup.
• May be set up to send notification e-mail if collected images show an image collection
problem has occurred. Allows confidence that unattended data collection is occurring
as desired.
• Maximum length of experiment determined by specimen thickness, image capture time
and number of images collected.
• Recently acquired data can be viewed without pausing acquisition.
• Unattended, once setup.
• May be set up to send notification e-mail if collected images show an image collection
problem has occurred. Allows confidence that unattended data collection is occurring
as desired.
• Maximum length of experiment determined by specimen thickness, image capture time
and number of images collected.
• Recently acquired data can be viewed without pausing acquisition.
Typical specimen size and requirements
• Typical block face size: 600 um x 600 um.
• Embedding resin: Epon, Durcupan, or Araldite
• Contrast: en-bloc staining (heavy metals)
• Typical block face size: 600 um x 600 um.
• Embedding resin: Epon, Durcupan, or Araldite
• Contrast: en-bloc staining (heavy metals)
Imaging Modes
Single frame per cut
• Single frame per cut
• Multiple fields of view and magnifications per cut
• Stage Montage for large fields of view
Image throughput
• Theoretical maximum: 316 GB/day or 2.22 TB/week
• Sustained operation: 10-315 GB/day or 0.70-2.21 TB/week
• Theoretical maximum: 316 GB/day or 2.22 TB/week
• Sustained operation: 10-315 GB/day or 0.70-2.21 TB/week
Sample throughput
0.5 µm
Gatan Microscopy Suite® 2
3View®2 operates on the new Gatan Microscopy Suite® (GMS) 2 64-bit platform with 12-96 GB of available memory. In
addition 3View®2XP now has three imaging modes, single image, montage, and multi ROI. The single image acquisition is
the classic 3View® mode with an improved interface. The new montage feature sets up an automated volume acquisition of
adjacent area allowing the images to be stitched together creating a super-volume. Multi ROI allows the user to image
several different areas specifying image size, pixel size, and dwell times for each individual ROI.
3View®2
1 min
Acquisition Cycle
20 sec
Acquisition Cycle
3View 2
®
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
30
50 200
43 x 43 x 43
72 x 72 x 72
288 x 288 x 288
302 x 302 x 302
525 x 525 x 525
600 x 600 x 600 (3 Days)
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
30
50 200
130 x 130 x 130
216 x 216 x 216
864 x 864 x 864
600 x 600 x 600 (6 Days)
600 x 600 x 600 (4 Days)
600 x 600 x 600 (1 Day)
Serial Block Face Imaging
500 nm
1 min
Acquisition Cycle
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
15
50 200
21 x 21 x 21
72 x 72 x 72
288 x 288 x 288
151 x 151 x 151
525 x 525 x 525
600 x 600 x 600 (3 Days)
20 sec
Acquisition Cycle
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
15
50 200
64 x 64 x 64
216 x 216 x 216
864 x 864 x 864
453 x 453 x 453
600 x 600 x 600 (4 Days)
600 x 600 x 600 (1 Day)
SEM Port Requirements
• One small port required for BSED feed through.
Ordering
Please contact your local Gatan Sales office for price and ordering information. Worldwide Sales contact information is available at www.gatan.com/company/sales.
• One small port required for BSED feed through.
Specifications and recommendations are subject to change.
250 nm
[email protected]
5794 W. Las Positas Blvd.
Pleasanton, CA 94588 USA
Corporate Office 925.463.0200
Worldwide Sales Offices:
www.gatan.com
BR-3View®2-FL1-CA-NOV11
Western USA Sales 925.463.0200
Eastern USA Sales 724.776.5260
UK 44.1235.540160
Germany 49.89.358084.0
France 33.1.69.11.03.69
Japan 81.3.5639.2772
Singapore 1.65.6408.6230
Cover Image: Top: Ultra resolution region of a segmented dendrite in green with synapses in yellow,
and synaptic vesicles in red. Lower left: Selected serial images with the reconstructed dendrite in green.
Lower right: Single SBFSEM image of a dendritic spine, synapse and synaptic vesicles. Images generated
by Gatan 3View®2. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for
Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented
using Imaris to create a 3D model of a neuron of interest.
3View® is a product based on work performed by W. Denk and H. Horstmann, Max-Planck Institute for Medical Research, Heidelberg, Germany. “Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure”. Plos Biology, 2004.2(11):p. 1900–1909.
1
This publication is the copyright of Gatan, Inc. and contains information that may not be used or reproduced unless
agreed by the company in writing. Specifications, recommendations and other technical data are subject to change.
Please consult your local Gatan Sales office for current information. 3View®, 3View®2, 3View®2XP, DigitalMicrograph®,
and Gatan Microscopy Suite® are trademarks of Gatan, Inc. © Gatan, Inc. 2011. All rights reserved.
ANALYTICAL TEM DIGITAL IMAGING SPECIMEN PREPARATION TEM SPECIMEN HOLDERS SEM PRODUCTS SOFTWARE
Serial
Block
Face
Imaging
EM Resolution to Ultra Resolution
The Gatan 3View®2 offers life science researchers the ability to
obtain in situ 3D data at remarkably fine depth resolution by
operating a high-precision ultramicrotome within a field
emission gun scanning electron microscope (FEGSEM). Based
on the groundbreaking serial block-face scanning electron
microscopy (SBFSEM) work performed by MPI Heidelberg1, the
revolutionary 3View®2 allows automated acquisition of 3D
ultrastructure by sequentially imaging a freshly cut, resinembedded block face. Unlike the 3D imaging techniques in
light microscopy, the spatial resolution provided by 3View®2 in
the Z direction is comparable to its X-Y resolution and does not
degrade with depth.
Complement Confocal
Main image: A 3D reconstruction of a dendrite from a 15,625 μm³ (25 x 25 x 25 μm) volumetric data set
containing 500 serial images of mouse cerebellum generated by Gatan 3View®2. Dendrite structure (green),
buttons (yellow), and vesicles (red). Inset images, clockwise from top left: Confocal image of a dendrite. Wire
frame traces rendered into a volumetric model. Ultra resolution dendritic spine model with synapses. 3View®2
image showing wire frame traces. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center
for Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented using
Imaris to create a 3D model of a neuron of interest.
Life Science
Top left: High resolution, mouse kidney, 8192 x 8192 pixel image acquired with 1.5 nm pixels. Top middle: Large
field of view, mouse kidney, 8192 x 8192 pixel image acquired with 80 nm pixels. Right: C. elegans prepared by
high pressure freezing; 4096 x 4096 pixel image acquired with 25 nm pixels; sample courtesy of Kent McDonald,
University of California, Berkeley. Bottom left: Mouse sciatic nerve, 2048 x 2048 pixel image acquired with 5 nm
pixels. Bottom middle: 3D visualization of mouse sciatic nerve axons. 3D data set contains 1,000 2048 x 2048
serial images collected with 5 nm pixels and segmented using the program Imaris. Sample courtesy of Gabrial
Corfas, Harvard University and Children’s Hospital Boston. Images generated by Gatan 3View®2.
Introduction
3View®2 is an automated sectioning and image capture system for the VP-FESEM. The purpose of the 3View®2 is
to collect serial images from a embedded sample unattended. The result produces a stack of aligned images which
can be viewed in sequence or further processed using 3D reconstruction and measurement software.
Sample Preparation
A biological sample needs to be prepared similar to the preparation used for the microtome or TEM. The sample
must be fixed, stained and embedded. See the sample preparation note for further details. The reason for the
preparation is that the sample must hold up to hours of imaging in the FESEM. This is why it must be fixed and
embedded in resin or epoxy. Since the specimen block is viewed using Gatan’s backscatter detector, the staining is
necessary for contrast.
Operation
The 3View®2 setup and operation is simple. The system is provided on its own chamber door that fits a specific
microscope. The standard stage is removed from the FESEM by venting the chamber. 3View®2 replaces the
standard stage, and the sample is loaded into the 3View®2.
500 nm
Image of Hela cells, stably expressing LC-GFP grown on gridded glass bottom coverslips dishes, starved for 2
hours in serum-free medium and cells of interest identified by confocal microscopy. The cells were then processed
in-situ for electron microscopy and the coverslips dissolved from the epoxy resin with hydrofluoric acid. The cells
were again identified in the resin block and in subsequent serial images generated by Gatan 3View®2. Samples
courtesy of David Dinsdale, MRC Toxicology Unit, University of Leicester, UK.
Materials Science
Upper and lower left: Images of anodized coating on aluminum surface generated by Gatan 3View®2. Sample
and data provided courtesy of Teruo Hashimoto and George Thompson, The University of Manchester, United
Kingdom. Middle: 3D visualization of aluminum alloy with manganese particles generated by Gatan 3View®2.
3D dataset contains 1,000 1024 x 1024 serial images with a pixel size of 15 nm and a cut thickness of 15 nm.
3D model created in DigitalMicrograph® using the 3DVisualization plugin.
The operator can choose a number of settings in set up of the unattended run. The cut step size can be set from 5
to 200 nanometers. The speed of the diamond knife (cutting speed) can be set from 0.05 to 5.0 mm/sec. The
number of images to be collected is set by the operator. This depends on how much material one wants to section
through. The 3View®2 can traverse through 600 microns in depth (Z).
Imaging
The images are acquired from the block-face, not the sections which are discarded. Image capture is done using a
Gatan backscatter system that is optimized for high signal collection at low accelerating voltage. Because the
sample is embedded in a nonconductive resin or epoxy, sample charging is almost inevitable. This is best handled
using variable pressure or charge compensation (such as gas injector) and low accelerating voltage. The operator
has a choice of image(s) to be collected after each cut. One can just take one image per cut or many. Those
images can be at several magnifications or a series of images such as a montage.
3View 2
®
Serial
Block
Face
Imaging
EM Resolution to Ultra Resolution
The Gatan 3View®2 offers life science researchers the ability to
obtain in situ 3D data at remarkably fine depth resolution by
operating a high-precision ultramicrotome within a field
emission gun scanning electron microscope (FEGSEM). Based
on the groundbreaking serial block-face scanning electron
microscopy (SBFSEM) work performed by MPI Heidelberg1, the
revolutionary 3View®2 allows automated acquisition of 3D
ultrastructure by sequentially imaging a freshly cut, resinembedded block face. Unlike the 3D imaging techniques in
light microscopy, the spatial resolution provided by 3View®2 in
the Z direction is comparable to its X-Y resolution and does not
degrade with depth.
Complement Confocal
Main image: A 3D reconstruction of a dendrite from a 15,625 μm³ (25 x 25 x 25 μm) volumetric data set
containing 500 serial images of mouse cerebellum generated by Gatan 3View®2. Dendrite structure (green),
buttons (yellow), and vesicles (red). Inset images, clockwise from top left: Confocal image of a dendrite. Wire
frame traces rendered into a volumetric model. Ultra resolution dendritic spine model with synapses. 3View®2
image showing wire frame traces. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center
for Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented using
Imaris to create a 3D model of a neuron of interest.
Life Science
Top left: High resolution, mouse kidney, 8192 x 8192 pixel image acquired with 1.5 nm pixels. Top middle: Large
field of view, mouse kidney, 8192 x 8192 pixel image acquired with 80 nm pixels. Right: C. elegans prepared by
high pressure freezing; 4096 x 4096 pixel image acquired with 25 nm pixels; sample courtesy of Kent McDonald,
University of California, Berkeley. Bottom left: Mouse sciatic nerve, 2048 x 2048 pixel image acquired with 5 nm
pixels. Bottom middle: 3D visualization of mouse sciatic nerve axons. 3D data set contains 1,000 2048 x 2048
serial images collected with 5 nm pixels and segmented using the program Imaris. Sample courtesy of Gabrial
Corfas, Harvard University and Children’s Hospital Boston. Images generated by Gatan 3View®2.
Introduction
3View®2 is an automated sectioning and image capture system for the VP-FESEM. The purpose of the 3View®2 is
to collect serial images from a embedded sample unattended. The result produces a stack of aligned images which
can be viewed in sequence or further processed using 3D reconstruction and measurement software.
Sample Preparation
A biological sample needs to be prepared similar to the preparation used for the microtome or TEM. The sample
must be fixed, stained and embedded. See the sample preparation note for further details. The reason for the
preparation is that the sample must hold up to hours of imaging in the FESEM. This is why it must be fixed and
embedded in resin or epoxy. Since the specimen block is viewed using Gatan’s backscatter detector, the staining is
necessary for contrast.
Operation
The 3View®2 setup and operation is simple. The system is provided on its own chamber door that fits a specific
microscope. The standard stage is removed from the FESEM by venting the chamber. 3View®2 replaces the
standard stage, and the sample is loaded into the 3View®2.
500 nm
Image of Hela cells, stably expressing LC-GFP grown on gridded glass bottom coverslips dishes, starved for 2
hours in serum-free medium and cells of interest identified by confocal microscopy. The cells were then processed
in-situ for electron microscopy and the coverslips dissolved from the epoxy resin with hydrofluoric acid. The cells
were again identified in the resin block and in subsequent serial images generated by Gatan 3View®2. Samples
courtesy of David Dinsdale, MRC Toxicology Unit, University of Leicester, UK.
Materials Science
Upper and lower left: Images of anodized coating on aluminum surface generated by Gatan 3View®2. Sample
and data provided courtesy of Teruo Hashimoto and George Thompson, The University of Manchester, United
Kingdom. Middle: 3D visualization of aluminum alloy with manganese particles generated by Gatan 3View®2.
3D dataset contains 1,000 1024 x 1024 serial images with a pixel size of 15 nm and a cut thickness of 15 nm.
3D model created in DigitalMicrograph® using the 3DVisualization plugin.
The operator can choose a number of settings in set up of the unattended run. The cut step size can be set from 5
to 200 nanometers. The speed of the diamond knife (cutting speed) can be set from 0.05 to 5.0 mm/sec. The
number of images to be collected is set by the operator. This depends on how much material one wants to section
through. The 3View®2 can traverse through 600 microns in depth (Z).
Imaging
The images are acquired from the block-face, not the sections which are discarded. Image capture is done using a
Gatan backscatter system that is optimized for high signal collection at low accelerating voltage. Because the
sample is embedded in a nonconductive resin or epoxy, sample charging is almost inevitable. This is best handled
using variable pressure or charge compensation (such as gas injector) and low accelerating voltage. The operator
has a choice of image(s) to be collected after each cut. One can just take one image per cut or many. Those
images can be at several magnifications or a series of images such as a montage.
3View 2
®
Serial
Block
Face
Imaging
EM Resolution to Ultra Resolution
The Gatan 3View®2 offers life science researchers the ability to
obtain in situ 3D data at remarkably fine depth resolution by
operating a high-precision ultramicrotome within a field
emission gun scanning electron microscope (FEGSEM). Based
on the groundbreaking serial block-face scanning electron
microscopy (SBFSEM) work performed by MPI Heidelberg1, the
revolutionary 3View®2 allows automated acquisition of 3D
ultrastructure by sequentially imaging a freshly cut, resinembedded block face. Unlike the 3D imaging techniques in
light microscopy, the spatial resolution provided by 3View®2 in
the Z direction is comparable to its X-Y resolution and does not
degrade with depth.
Complement Confocal
Main image: A 3D reconstruction of a dendrite from a 15,625 μm³ (25 x 25 x 25 μm) volumetric data set
containing 500 serial images of mouse cerebellum generated by Gatan 3View®2. Dendrite structure (green),
buttons (yellow), and vesicles (red). Inset images, clockwise from top left: Confocal image of a dendrite. Wire
frame traces rendered into a volumetric model. Ultra resolution dendritic spine model with synapses. 3View®2
image showing wire frame traces. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center
for Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented using
Imaris to create a 3D model of a neuron of interest.
Life Science
Top left: High resolution, mouse kidney, 8192 x 8192 pixel image acquired with 1.5 nm pixels. Top middle: Large
field of view, mouse kidney, 8192 x 8192 pixel image acquired with 80 nm pixels. Right: C. elegans prepared by
high pressure freezing; 4096 x 4096 pixel image acquired with 25 nm pixels; sample courtesy of Kent McDonald,
University of California, Berkeley. Bottom left: Mouse sciatic nerve, 2048 x 2048 pixel image acquired with 5 nm
pixels. Bottom middle: 3D visualization of mouse sciatic nerve axons. 3D data set contains 1,000 2048 x 2048
serial images collected with 5 nm pixels and segmented using the program Imaris. Sample courtesy of Gabrial
Corfas, Harvard University and Children’s Hospital Boston. Images generated by Gatan 3View®2.
Introduction
3View®2 is an automated sectioning and image capture system for the VP-FESEM. The purpose of the 3View®2 is
to collect serial images from a embedded sample unattended. The result produces a stack of aligned images which
can be viewed in sequence or further processed using 3D reconstruction and measurement software.
Sample Preparation
A biological sample needs to be prepared similar to the preparation used for the microtome or TEM. The sample
must be fixed, stained and embedded. See the sample preparation note for further details. The reason for the
preparation is that the sample must hold up to hours of imaging in the FESEM. This is why it must be fixed and
embedded in resin or epoxy. Since the specimen block is viewed using Gatan’s backscatter detector, the staining is
necessary for contrast.
Operation
The 3View®2 setup and operation is simple. The system is provided on its own chamber door that fits a specific
microscope. The standard stage is removed from the FESEM by venting the chamber. 3View®2 replaces the
standard stage, and the sample is loaded into the 3View®2.
500 nm
Image of Hela cells, stably expressing LC-GFP grown on gridded glass bottom coverslips dishes, starved for 2
hours in serum-free medium and cells of interest identified by confocal microscopy. The cells were then processed
in-situ for electron microscopy and the coverslips dissolved from the epoxy resin with hydrofluoric acid. The cells
were again identified in the resin block and in subsequent serial images generated by Gatan 3View®2. Samples
courtesy of David Dinsdale, MRC Toxicology Unit, University of Leicester, UK.
Materials Science
Upper and lower left: Images of anodized coating on aluminum surface generated by Gatan 3View®2. Sample
and data provided courtesy of Teruo Hashimoto and George Thompson, The University of Manchester, United
Kingdom. Middle: 3D visualization of aluminum alloy with manganese particles generated by Gatan 3View®2.
3D dataset contains 1,000 1024 x 1024 serial images with a pixel size of 15 nm and a cut thickness of 15 nm.
3D model created in DigitalMicrograph® using the 3DVisualization plugin.
The operator can choose a number of settings in set up of the unattended run. The cut step size can be set from 5
to 200 nanometers. The speed of the diamond knife (cutting speed) can be set from 0.05 to 5.0 mm/sec. The
number of images to be collected is set by the operator. This depends on how much material one wants to section
through. The 3View®2 can traverse through 600 microns in depth (Z).
Imaging
The images are acquired from the block-face, not the sections which are discarded. Image capture is done using a
Gatan backscatter system that is optimized for high signal collection at low accelerating voltage. Because the
sample is embedded in a nonconductive resin or epoxy, sample charging is almost inevitable. This is best handled
using variable pressure or charge compensation (such as gas injector) and low accelerating voltage. The operator
has a choice of image(s) to be collected after each cut. One can just take one image per cut or many. Those
images can be at several magnifications or a series of images such as a montage.
3View 2
®
3View 2
Features and Capabilities
Specifications, Recommendations and Ordering
®
Specification/
Recommendation
Simplifying EM
The 3View®2 setup and operation is intuitive and simple. Removing the most difficult
step, cutting and collecting ultrathin sections, 3View®2 can turn an embedded sample into
thousands on images overnight. In addition 3View®2 can also cut thinner than a conventional
ultramictotome, cutting a sample from <15 nm to 200 nanometers with a total traverse of
600 microns.
Cutting Thin
3View®2XP has the ability to cut biological and material samples thinner than 15 nm. This is made possible by precise z
stage control, stabilization of the microtome, and a custom diamond knife. The 3View®2XP z movement is controlled by a
piezoelectric device, allowing precise movements at the nanometer scale. Adjusting the cutting thickness is controlled by
DigitalMicrograph®. In addition, the 3View®2XP stage has been stabilized to minimize vibrations reducing cutting discrepancy
thickness. Image: Mouse kidney prepared with the National Center for Microscopy and Imaging Research (NCMIR)
3View®2XP specimen preparation protocol embedded with Durcupan resin imaged with 7 nm pixels, and cut with 7 nm cuts
creating 7 nm isotropic voxels.
250 nm
3View®2XP
Recommended SEM
• Variable Pressure, Field Emission Gun SEM with a large chamber (Call Gatan for
specific SEM compatibility). A FEG or LaB6 is preferred due to long term beam
stability and brightness.
• Field Emission Gun SEM with a large chamber (Call Gatan for specific SEM compatibility).
A FEG is preferred due to long term beam stability & brightness, allowing cutting runs
on the order of 10 days or more.
Charge Neutralization
• Variable pressure SEM required; 5-40 Pa is typical at 2 kV.
• Variable pressure SEM is recommended but not required; 5-40 Pa is typical at 2 kV.
Accelerating Voltage
• 1 kV – 5 kV
• <1 kV – 5 kV
Cutting speed
• User defined: 0.1-1.2 mm/sec
• Recommended speed: 0.5-1 mm/sec
• User defined: 0.1-1.2 mm/sec
• Recommended speed: 0.5-1 mm/sec
Cut thickness
• Microtome can cut from 30 to 200 nm. 30 to 50 nm is typical with biological specimens.
• Microtome can cut from 15 to 200 nm. 25 to 50 nm is typical with biological specimens.
Knife Cutting Travel Distance
• 1.2 mm
• 1.2 mm
Z travel distance
• Maximum of 600 µm
• Maximum of 600 µm
3View® stage travel distance
• Traverses approximately +/-700 µm in X and Y.
• Traverses approximately +/-700 µm in X and Y.
SEM stage
• 3View®2 replaces SEM stage. High Stability Manual x-y- stage (movement sufficient to
cover 1 mm x 1 mm specimen block).
• 3View®2XP replaces SEM stage. High Vacuum Compatible automated x-y- stage
(movement sufficient to cover 1 mm x 1 mm specimen block)
Working distance
• Approximately 6 mm when used with Gatan BSED.
• Approximately 6 mm when used with Gatan BSED.
Low magnification field of view
• Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is
typically 1.2 mm x 1.2 mm.
• Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is
typically 1.2 mm x 1.2 mm.
Image acquisition
• Gatan DigiScan® uses SEM external scan control input with Firewire.
• 2-16 bit analog inputs can work simultaneously. 3View®2 acquisition is typically BSED
only. Gatan recommends its own BSE detector optimized for low kV image collection.
• Gatan DigiScan® uses SEM external scan control input with Firewire.
• 2-16 bit analog inputs can work simultaneously. 3View®2XP acquisition is typically
BSED only. Gatan recommends its own BSE detector optimized for low kV
image collection.
DigiScan® pixel density
3View®2 supports images up to 24k x 32k pixels. Actual image size is SEM dependent.
3View®2XP supports images up to 24k x 32k pixels. Actual image size is SEM dependent.
Pixel dwell time
• Microscope and sample dependant. 1-20 µs is typical.
• Microscope and sample dependant. 1-20 µs is typical.
Image scanning
NA
• Choice of single pass or configurable multiple frames.
Image calibration
• DigiScan® calibration is kV and magnification specific.
• DigiScan® calibration is kV and magnification specific.
SEM-PC communication
• Follows SEM protocol. Communication for kV, FOV, Magnification, Vacuum,
Beam Blanking.
• Follows SEM protocol. Communication for kV, FOV, Magnification, Vacuum,
Beam Blanking.
3View® setup
• Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom
microscope with chamber door at air.
• Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom
microscope with chamber door at air.
3View® operation
• Unattended, once setup.
• May be set up to send notification e-mail if collected images show an image collection
problem has occurred. Allows confidence that unattended data collection is occurring
as desired.
• Maximum length of experiment determined by specimen thickness, image capture time
and number of images collected.
• Recently acquired data can be viewed without pausing acquisition.
• Unattended, once setup.
• May be set up to send notification e-mail if collected images show an image collection
problem has occurred. Allows confidence that unattended data collection is occurring
as desired.
• Maximum length of experiment determined by specimen thickness, image capture time
and number of images collected.
• Recently acquired data can be viewed without pausing acquisition.
Typical specimen size and requirements
• Typical block face size: 600 um x 600 um.
• Embedding resin: Epon, Durcupan, or Araldite
• Contrast: en-bloc staining (heavy metals)
• Typical block face size: 600 um x 600 um.
• Embedding resin: Epon, Durcupan, or Araldite
• Contrast: en-bloc staining (heavy metals)
Imaging Modes
Single frame per cut
• Single frame per cut
• Multiple fields of view and magnifications per cut
• Stage Montage for large fields of view
Image throughput
• Theoretical maximum: 316 GB/day or 2.22 TB/week
• Sustained operation: 10-315 GB/day or 0.70-2.21 TB/week
• Theoretical maximum: 316 GB/day or 2.22 TB/week
• Sustained operation: 10-315 GB/day or 0.70-2.21 TB/week
Sample throughput
0.5 µm
Gatan Microscopy Suite® 2
3View®2 operates on the new Gatan Microscopy Suite® (GMS) 2 64-bit platform with 12-96 GB of available memory. In
addition 3View®2XP now has three imaging modes, single image, montage, and multi ROI. The single image acquisition is
the classic 3View® mode with an improved interface. The new montage feature sets up an automated volume acquisition of
adjacent area allowing the images to be stitched together creating a super-volume. Multi ROI allows the user to image
several different areas specifying image size, pixel size, and dwell times for each individual ROI.
3View®2
1 min
Acquisition Cycle
20 sec
Acquisition Cycle
3View 2
®
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
30
50 200
43 x 43 x 43
72 x 72 x 72
288 x 288 x 288
302 x 302 x 302
525 x 525 x 525
600 x 600 x 600 (3 Days)
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
30
50 200
130 x 130 x 130
216 x 216 x 216
864 x 864 x 864
600 x 600 x 600 (6 Days)
600 x 600 x 600 (4 Days)
600 x 600 x 600 (1 Day)
Serial Block Face Imaging
500 nm
1 min
Acquisition Cycle
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
15
50 200
21 x 21 x 21
72 x 72 x 72
288 x 288 x 288
151 x 151 x 151
525 x 525 x 525
600 x 600 x 600 (3 Days)
20 sec
Acquisition Cycle
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
15
50 200
64 x 64 x 64
216 x 216 x 216
864 x 864 x 864
453 x 453 x 453
600 x 600 x 600 (4 Days)
600 x 600 x 600 (1 Day)
SEM Port Requirements
• One small port required for BSED feed through.
Ordering
Please contact your local Gatan Sales office for price and ordering information. Worldwide Sales contact information is available at www.gatan.com/company/sales.
• One small port required for BSED feed through.
Specifications and recommendations are subject to change.
250 nm
[email protected]
5794 W. Las Positas Blvd.
Pleasanton, CA 94588 USA
Corporate Office 925.463.0200
Worldwide Sales Offices:
www.gatan.com
BR-3View®2-FL1-CA-NOV11
Western USA Sales 925.463.0200
Eastern USA Sales 724.776.5260
UK 44.1235.540160
Germany 49.89.358084.0
France 33.1.69.11.03.69
Japan 81.3.5639.2772
Singapore 1.65.6408.6230
Cover Image: Top: Ultra resolution region of a segmented dendrite in green with synapses in yellow,
and synaptic vesicles in red. Lower left: Selected serial images with the reconstructed dendrite in green.
Lower right: Single SBFSEM image of a dendritic spine, synapse and synaptic vesicles. Images generated
by Gatan 3View®2. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for
Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented
using Imaris to create a 3D model of a neuron of interest.
3View® is a product based on work performed by W. Denk and H. Horstmann, Max-Planck Institute for Medical Research, Heidelberg, Germany. “Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure”. Plos Biology, 2004.2(11):p. 1900–1909.
1
This publication is the copyright of Gatan, Inc. and contains information that may not be used or reproduced unless
agreed by the company in writing. Specifications, recommendations and other technical data are subject to change.
Please consult your local Gatan Sales office for current information. 3View®, 3View®2, 3View®2XP, DigitalMicrograph®,
and Gatan Microscopy Suite® are trademarks of Gatan, Inc. © Gatan, Inc. 2011. All rights reserved.
ANALYTICAL TEM DIGITAL IMAGING SPECIMEN PREPARATION TEM SPECIMEN HOLDERS SEM PRODUCTS SOFTWARE
3View 2
Features and Capabilities
Specifications, Recommendations and Ordering
®
Specification/
Recommendation
Simplifying EM
The 3View®2 setup and operation is intuitive and simple. Removing the most difficult
step, cutting and collecting ultrathin sections, 3View®2 can turn an embedded sample into
thousands on images overnight. In addition 3View®2 can also cut thinner than a conventional
ultramictotome, cutting a sample from <15 nm to 200 nanometers with a total traverse of
600 microns.
Cutting Thin
3View®2XP has the ability to cut biological and material samples thinner than 15 nm. This is made possible by precise z
stage control, stabilization of the microtome, and a custom diamond knife. The 3View®2XP z movement is controlled by a
piezoelectric device, allowing precise movements at the nanometer scale. Adjusting the cutting thickness is controlled by
DigitalMicrograph®. In addition, the 3View®2XP stage has been stabilized to minimize vibrations reducing cutting discrepancy
thickness. Image: Mouse kidney prepared with the National Center for Microscopy and Imaging Research (NCMIR)
3View®2XP specimen preparation protocol embedded with Durcupan resin imaged with 7 nm pixels, and cut with 7 nm cuts
creating 7 nm isotropic voxels.
250 nm
3View®2XP
Recommended SEM
• Variable Pressure, Field Emission Gun SEM with a large chamber (Call Gatan for
specific SEM compatibility). A FEG or LaB6 is preferred due to long term beam
stability and brightness.
• Field Emission Gun SEM with a large chamber (Call Gatan for specific SEM compatibility).
A FEG is preferred due to long term beam stability & brightness, allowing cutting runs
on the order of 10 days or more.
Charge Neutralization
• Variable pressure SEM required; 5-40 Pa is typical at 2 kV.
• Variable pressure SEM is recommended but not required; 5-40 Pa is typical at 2 kV.
Accelerating Voltage
• 1 kV – 5 kV
• <1 kV – 5 kV
Cutting speed
• User defined: 0.1-1.2 mm/sec
• Recommended speed: 0.5-1 mm/sec
• User defined: 0.1-1.2 mm/sec
• Recommended speed: 0.5-1 mm/sec
Cut thickness
• Microtome can cut from 30 to 200 nm. 30 to 50 nm is typical with biological specimens.
• Microtome can cut from 15 to 200 nm. 25 to 50 nm is typical with biological specimens.
Knife Cutting Travel Distance
• 1.2 mm
• 1.2 mm
Z travel distance
• Maximum of 600 µm
• Maximum of 600 µm
3View® stage travel distance
• Traverses approximately +/-700 µm in X and Y.
• Traverses approximately +/-700 µm in X and Y.
SEM stage
• 3View®2 replaces SEM stage. High Stability Manual x-y- stage (movement sufficient to
cover 1 mm x 1 mm specimen block).
• 3View®2XP replaces SEM stage. High Vacuum Compatible automated x-y- stage
(movement sufficient to cover 1 mm x 1 mm specimen block)
Working distance
• Approximately 6 mm when used with Gatan BSED.
• Approximately 6 mm when used with Gatan BSED.
Low magnification field of view
• Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is
typically 1.2 mm x 1.2 mm.
• Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is
typically 1.2 mm x 1.2 mm.
Image acquisition
• Gatan DigiScan® uses SEM external scan control input with Firewire.
• 2-16 bit analog inputs can work simultaneously. 3View®2 acquisition is typically BSED
only. Gatan recommends its own BSE detector optimized for low kV image collection.
• Gatan DigiScan® uses SEM external scan control input with Firewire.
• 2-16 bit analog inputs can work simultaneously. 3View®2XP acquisition is typically
BSED only. Gatan recommends its own BSE detector optimized for low kV
image collection.
DigiScan® pixel density
3View®2 supports images up to 24k x 32k pixels. Actual image size is SEM dependent.
3View®2XP supports images up to 24k x 32k pixels. Actual image size is SEM dependent.
Pixel dwell time
• Microscope and sample dependant. 1-20 µs is typical.
• Microscope and sample dependant. 1-20 µs is typical.
Image scanning
NA
• Choice of single pass or configurable multiple frames.
Image calibration
• DigiScan® calibration is kV and magnification specific.
• DigiScan® calibration is kV and magnification specific.
SEM-PC communication
• Follows SEM protocol. Communication for kV, FOV, Magnification, Vacuum,
Beam Blanking.
• Follows SEM protocol. Communication for kV, FOV, Magnification, Vacuum,
Beam Blanking.
3View® setup
• Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom
microscope with chamber door at air.
• Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom
microscope with chamber door at air.
3View® operation
• Unattended, once setup.
• May be set up to send notification e-mail if collected images show an image collection
problem has occurred. Allows confidence that unattended data collection is occurring
as desired.
• Maximum length of experiment determined by specimen thickness, image capture time
and number of images collected.
• Recently acquired data can be viewed without pausing acquisition.
• Unattended, once setup.
• May be set up to send notification e-mail if collected images show an image collection
problem has occurred. Allows confidence that unattended data collection is occurring
as desired.
• Maximum length of experiment determined by specimen thickness, image capture time
and number of images collected.
• Recently acquired data can be viewed without pausing acquisition.
Typical specimen size and requirements
• Typical block face size: 600 um x 600 um.
• Embedding resin: Epon, Durcupan, or Araldite
• Contrast: en-bloc staining (heavy metals)
• Typical block face size: 600 um x 600 um.
• Embedding resin: Epon, Durcupan, or Araldite
• Contrast: en-bloc staining (heavy metals)
Imaging Modes
Single frame per cut
• Single frame per cut
• Multiple fields of view and magnifications per cut
• Stage Montage for large fields of view
Image throughput
• Theoretical maximum: 316 GB/day or 2.22 TB/week
• Sustained operation: 10-315 GB/day or 0.70-2.21 TB/week
• Theoretical maximum: 316 GB/day or 2.22 TB/week
• Sustained operation: 10-315 GB/day or 0.70-2.21 TB/week
Sample throughput
0.5 µm
Gatan Microscopy Suite® 2
3View®2 operates on the new Gatan Microscopy Suite® (GMS) 2 64-bit platform with 12-96 GB of available memory. In
addition 3View®2XP now has three imaging modes, single image, montage, and multi ROI. The single image acquisition is
the classic 3View® mode with an improved interface. The new montage feature sets up an automated volume acquisition of
adjacent area allowing the images to be stitched together creating a super-volume. Multi ROI allows the user to image
several different areas specifying image size, pixel size, and dwell times for each individual ROI.
3View®2
1 min
Acquisition Cycle
20 sec
Acquisition Cycle
3View 2
®
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
30
50 200
43 x 43 x 43
72 x 72 x 72
288 x 288 x 288
302 x 302 x 302
525 x 525 x 525
600 x 600 x 600 (3 Days)
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
30
50 200
130 x 130 x 130
216 x 216 x 216
864 x 864 x 864
600 x 600 x 600 (6 Days)
600 x 600 x 600 (4 Days)
600 x 600 x 600 (1 Day)
Serial Block Face Imaging
500 nm
1 min
Acquisition Cycle
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
15
50 200
21 x 21 x 21
72 x 72 x 72
288 x 288 x 288
151 x 151 x 151
525 x 525 x 525
600 x 600 x 600 (3 Days)
20 sec
Acquisition Cycle
Isotropic Voxel
Size (nm)
1 Day (µm)
1 Week (µm)
15
50 200
64 x 64 x 64
216 x 216 x 216
864 x 864 x 864
453 x 453 x 453
600 x 600 x 600 (4 Days)
600 x 600 x 600 (1 Day)
SEM Port Requirements
• One small port required for BSED feed through.
Ordering
Please contact your local Gatan Sales office for price and ordering information. Worldwide Sales contact information is available at www.gatan.com/company/sales.
• One small port required for BSED feed through.
Specifications and recommendations are subject to change.
250 nm
[email protected]
5794 W. Las Positas Blvd.
Pleasanton, CA 94588 USA
Corporate Office 925.463.0200
Worldwide Sales Offices:
www.gatan.com
BR-3View®2-FL1-CA-NOV11
Western USA Sales 925.463.0200
Eastern USA Sales 724.776.5260
UK 44.1235.540160
Germany 49.89.358084.0
France 33.1.69.11.03.69
Japan 81.3.5639.2772
Singapore 1.65.6408.6230
Cover Image: Top: Ultra resolution region of a segmented dendrite in green with synapses in yellow,
and synaptic vesicles in red. Lower left: Selected serial images with the reconstructed dendrite in green.
Lower right: Single SBFSEM image of a dendritic spine, synapse and synaptic vesicles. Images generated
by Gatan 3View®2. Sample courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for
Microscopy and Imaging Research, University of California, San Diego. Serial images were segmented
using Imaris to create a 3D model of a neuron of interest.
3View® is a product based on work performed by W. Denk and H. Horstmann, Max-Planck Institute for Medical Research, Heidelberg, Germany. “Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure”. Plos Biology, 2004.2(11):p. 1900–1909.
1
This publication is the copyright of Gatan, Inc. and contains information that may not be used or reproduced unless
agreed by the company in writing. Specifications, recommendations and other technical data are subject to change.
Please consult your local Gatan Sales office for current information. 3View®, 3View®2, 3View®2XP, DigitalMicrograph®,
and Gatan Microscopy Suite® are trademarks of Gatan, Inc. © Gatan, Inc. 2011. All rights reserved.
ANALYTICAL TEM DIGITAL IMAGING SPECIMEN PREPARATION TEM SPECIMEN HOLDERS SEM PRODUCTS SOFTWARE