Electron Microscopy Services
- List Electron Microscopy Services Here
- List Electron Microscopy Services Here
- List Electron Microscopy Services Here
Electron Microscopy Image Analysis
EM images are particularly challenging to segment because the objects of interest are defined only by edges, rather than by intensities. Still, moderately automated segmentation is possible. This example shows an automatic segmentation of membranes (red) and mitochondria (blue) in a slice of a FIB-SEM data set.
Electron Microscopy Sample Prep
CRITICAL POINT DRYING AND SPUTTER COATING FOR SEM
Prior to conventional SEM imaging, the sample must be carefully dehydrated and coated with a layer of heavy metal, often gold, only a few atoms thick. The dehydration is best performed using a device called a critical point dryer, which replaces water with miscible alcohol and then liquid CO2. Finally, the CO2 is evaporated under pressure at its critical point. This process avoids surface tension forces that can otherwise destroy ultrastructural details. Next, the dry sample is placed in a high vacuum coater in order to coat it with a layer of (conductive) metal only a few atoms thick. The coating is achieved by resistively heating the metal in a moderate vacuum until it evaporates. Some of the atoms then condense onto the sample.
SAMPLE PREPARATION FOR FOCUSED ION BEAM-SEM
FIB-SEM is a block face SEM imaging method that uses an ion beam to iteratively mill the block surface, creating a 3D image stack. Sample prep for FIB-SEM is similar to the methods used for TEM since the goal is to visualize internal structures based on back-scattered electrons. Following standard protocol, sample is fixed in glutaraldehyde and stained with osmium and other heavy metals. It is then embedded in Durcupan resin, which well-infiltrates that sample and is most compatible with ion beam milling. Before final imaging, which is practically limited to a very small region (~4 x 4 x 2 um), the resin block must be manually trimmed to find the desired region, which can be a very painstaking process.
ULTRAMICROTOMY AND ARRAY TOMOGRAPHY
Conventional TEM is based on imaging electrons that transmit through the sample. Thus, the sample must be very thin, in the range of 50-100 nm. Larger samples must first be fixed, stained with osmium, and then embedded in an epoxy resin. The resin block is then manually trimmed to find the region of interest and finally the desired region is sliced into ultra-thin sections for imaging.
In a technique called array tomography, a large sequence of hundreds of serial sections are cut from a resin block and placed in order (‘arrayed’) onto support. The sections are then imaged in an SEM and the many 2D images from each section are digitally reassembled into a single 3D image. This method is also useful for super-resolution light microscopy.
PROCESSING FOR CONVENTIONAL TEM
Biological samples must be strongly fixed, embedded in a resin for cutting, and stained with heavy metals prior to imaging with electrons in a high vacuum. The core can work with a range of resins and stains, depending on the sample. Macro-molecular samples can also be cast into a heavy metal salt and their imprint imaged, a process called negative staining. Our standard methods and protocols can be found here:
Protocol for TEM of suspensions
HIGH-PRESSURE FREEZING – FREEZE SUBSTITUTION
For very high-resolution TEM imaging where ultrastructural details are critical or to capture transient features that is perturbed by chemical fixation, an alternative is to freeze the sample very rapidly (<1 msec), in vitreous ice. This rapid freezing is accomplished in a high-pressure freezer. After freezing, the sample is then chemically fixed and embedded while still frozen using a process called freeze-substitution. Finally, the sample is brought back to room temperature for imaging. Alternatively, high pressure freezing is used to prepare tissue or larger cells for cryo-electron microscopy. In this case, the sample is kept frozen in ice throughout the imaging process.
TOKUYASU METHOD FOR IMMUNOGOLD LABELING
Immunogold labeling is an antibody labeling method for electron microscopy, where small (5 nm) gold particles are used for contrast. Due to ease of use, we recommend to first attempt immunogold labeling using post-embed staining of acrylic resin sections (LR White). Though less technically challenging to perform, post-resin embed staining often does not work very well because the antigen may be damaged during the fixation/embedding process or antibody may not be able to access the antigen. An alternative approach is to infiltrate the sample with a saturated sucrose solution and then freeze it in liquid nitrogen. While still frozen, ultrathin sections are cut on a cryo-ultramicrotome. The sections are then immunostained prior to embedding. Cutting frozen sections is extremely challenging, but when successful the immunostaining steps are much more likely to work well.
BRCF Electron Microscopy Equipment
JEOL JEM 1400 plus LaB6 TEM
Location: Microscopy Core; BSRB A825
The TEM uses a lanthanum hexaboride filament (LaB6) to generate the electron beam, which ~ 10 X brighter than a tungsten (W) filament. The resolution is 0.38nm with point to point and 0.2nm with lattice at 40 – 120 keV. The TEM is equipped with an AMT NanoSprint 12 scientific-grade CMOS camera, which achieves high sensitivity and promotes high-speed TEM imaging. This sensor offers a large 12-megapixel sampling region while maintaining 55 frames per second readout. Quick Start Manual.
Zeiss EVO 15 LaB6 SEM
Location: Microscopy Core; BSRB A821
The SEM uses a lanthanum hexaboride filament (LaB6) to generate the electron beam, which ~ 10 X brighter than a tungsten (W) filament. The resolution is 3nm at 30 keV SE and 8nm at 3 keV with high vacuum secondary electron. Available detectors are secondary electron (SE) – Everhart-Thornley detector, HDBSD – high definition 4 quadrant backscattered electron detector, C2D – cascade current detector in variable pressure, and STEM – scanning transmission electron microscopy detector. The image can achieve 32,000 x 24,000 pixels with single acquisition by integration and averaging. SmartSEM Manual… Coming soon.
Equipment Available from the MICHIGAN CENTER FOR MATERIALS CHARACTERIZATION
JEOL 3100R05 TEM
Location: Michigan Center for Materials Characterization, NCRC BLDG 22 Rm G026
High resolution (>0.1 nm) TEM operated at 200 or 300 keV. Single or double tilt JEOL stage. Gatan Ultrascan 1000 CCD TV camera. This system is used for tilt tomography on 200-300 nm semi-thin resin sections. The Microscopy Core’s JEOL 1400plus is sufficient for most biological TEM applications.
Full-service TEM sample preparation is available.
TESCAN MIRA3 FIELD EMISSION SEM
Location: Michigan Center for Materials Characterization, NCRC BLDG 22 Rm G022
High resolution (>2 nm) field emission SEM operating at 0-30 keV accelerating voltages. Includes Everhart-Thornley secondary electron detector and a back-scattered electron detector. Motorized stage for xyz translation, rotation, and +/- 90 degree tilt. Used for non-routine, high-resolution SEM applications. The Microscopy Core’s bench top SEM is sufficient for most biological SEM applications.
Full-service SEM sample preparation is available.
THERMOFISHER HELIOS 650 NANOLAB FOCUSED ION BEAM SEM
Location: Michigan Center for Materials Characterization; NCRC BLDG 22 Rm G025
High resolution (>2 nm) field emission SEM operating at 0-30 keV accelerating voltages with Everhart-Thornley or Through-the-lens SE Detectors. Concentric backscatter detector. Gallium ion beam has 0.1-65 nA current with down to 4 nm milling resolution. Used for 3D FIB-SEM imaging of Durcupan resin blocks.
Full-service FIB-SEM sample preparation is available.
ZEISS XRADIA VERSA 520 X-RAY MICROSCOPE
Location: Michigan Center for Materials Characterization; NCRC BLDG 22 Rm G029
High resolution (>1 um) microCT with phase contrast optics and variable magnification with 30-160 keV beam. Useful for mineralized tissues or 3D overview imaging of EM resin blocks.