Serial Block Face Imaging

Transcription

1 3View 2 Serial Block Face Imaging 500 nm 250 nm ANALYTICAL TEM DIGITAL IMAGING SPECIMEN PREPARATION TEM SPECIMEN HOLDERS SEM PRODUCTS SOFTWARE

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 Heidelberg 1, 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. 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. 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.

3 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. 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.

4 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, 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. 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, 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. 3View 2

5 3View Features and Capabilities 2 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 0.5 µm Gatan Microscopy Suite 2 3View 2 operates on the new Gatan Microscopy Suite (GMS) 2 64-bit platform with 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.

6 Specifications, Recommendations and Ordering Specification/ Recommendation 3View 2 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: mm/sec Recommended speed: mm/sec User defined: mm/sec Recommended speed: 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 Image acquisition Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is typically 1.2 mm x 1.2 mm. Gatan DigiScan uses SEM external scan control input with Firewire 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. Depends on Specific electron optics. Gatan BSED has a 1 mm aperture and FOV is typically 1.2 mm x 1.2 mm. Gatan DigiScan uses SEM external scan control input with Firewire 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 µs is typical. Microscope and sample dependant 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 3View setup 3View operation Typical specimen size and requirements Follows SEM protocol. Communication for kv, FOV, Magnification, Vacuum, Beam Blanking. Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom microscope with chamber door at air. Unattended, once setup. May be set up to send notification 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 block face size: 600 um x 600 um. Embedding resin: Epon, Durcupan, or Araldite Contrast: en-bloc staining (heavy metals) Follows SEM protocol. Communication for kv, FOV, Magnification, Vacuum, Beam Blanking. Defined protocol for approach sequence protects diamond knife. Utilizes optical zoom microscope with chamber door at air. Unattended, once setup. May be set up to send notification 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 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: GB/day or TB/week Theoretical maximum: 316 GB/day or 2.22 TB/week Sustained operation: GB/day or TB/week Sample throughput Isotropic Voxel 1 Day (µm) 1 Week (µm) 1 min Acquisition Cycle x 43 x x 302 x x 72 x x 525 x x 288 x x 600 x 600 (3 Days) Isotropic Voxel 1 Day (µm) 1 Week (µm) 20 sec Acquisition Cycle x 130 x x 600 x 600 (6 Days) x 216 x x 600 x 600 (4 Days) x 864 x x 600 x 600 (1 Day) Isotropic Voxel 1 Day (µm) 1 Week (µm) 1 min Acquisition Cycle x 21 x x 151 x x 72 x x 525 x x 288 x x 600 x 600 (3 Days) Isotropic Voxel 1 Day (µm) 1 Week (µm) 20 sec Acquisition Cycle x 64 x x 453 x x 216 x x 600 x 600 (4 Days) x 864 x x 600 x 600 (1 Day) SEM Port Requirements One small port required for BSED feed through. 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 Specifications and recommendations are subject to change. info@gatan.com 5794 W. Las Positas Blvd. Pleasanton, CA USA Corporate Office Worldwide Sales Offices: Western USA Sales Eastern USA Sales UK Germany France Japan Singapore 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. 1 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, (11):p 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 All rights reserved. BR-3View 2-FL1-CA-NOV11