Scanning Electron Microscopy is one of the most valuable tools a cell biologist can utilize to examine the internal structure of cells. Samples must undergo complex preparation steps to withstand the low pressure and high-temperature environment inside the microscope.
This article details four methods to image internal structures of biological tissues and cells using SEM:
- Fix, dehydrate and embed in resin
- Rapid freeze, fracture and coat
- LS BSE Detection
Fix, Dehydrate and Embed in Resin
Of the various methods, one of the simplest is to fix a sample with a heavy metal stain, dehydrate it and set it into resin before imaging. As the resin is non-conductive, silver paint can be used to surround the sample before coating with gold. The block face is then imaged by mounting the resin on a special microtome and exposing a fresh area with a diamond knife.
Block face of lung tissue ~1mm across
Alternatively, a dermatome (knife) can be used to slice thin sections, which can be mounted in a sample holder or on tape, which in turn can be mounted onto a support and coated with gold or palladium before being viewed with transmission electron microscopy or more likely STEM (scanning transmission electron microscopy) because of its flexibility in having more imaging geometries.
STEM Bright Field Image of sectioned tissue
STEMs principal advantages over TEM is in enabling the use of other electron signals that cannot be spatially correlated in TEM, including secondary electrons, scattered beam electrons, characteristic X-rays, and electron energy loss. STEM usually requires thin sample sections and can use a variety of dark field imaging geometries and beam energies to provide different perspectives or contrasts on the sample to build a representative image. STEM also has advantages over conventional SEM as it provides greater spatial resolution.
Using these methods, extremely high-resolution images can be attained, for example in the below image, which is zoomed into a tissue sample, where nuclear membranes, mitochondria and other cellular structures can clearly be seen.
16k x 16k pixel image, 1.0us/pixel, 6nm pixels
Tescan also offers image Snapper software that allows these images to be stitched together to form panoramas such as the below taken using 36 16k x 16k images.
Large panorama of part of a single section
Rapid Freeze, Fracture and Coat
Freeze-fracturing, freeze-etch or freeze-and-break is a method used for examining the internal structures of cells in a similar method to the rapid freeze and coat methods used to image the outer surfaces of cells. Freezing is performed as quickly as possible to minimize the size of ice crystals, the sample is then transferred to a station to be fractured, etched and coated before being moved to the SEM.
A cry-fractured leaf
Freeze-fracture provides a way of opening up cells and tissues for an internal view of the cytoplasm and nucleus and can also provide an internal view of a cytoplasmic organelle if the plane of fracture cuts through the organelle.
Selective removal of soluble or other components may be necessary for a deeper view of the structure at the fracture face. This can be realized by osmium digestion, glycerol extraction, by delaying fixation until after freeze-fracture and thawing, or by prior treatment with detergent to remove cell membranes and wash out soluble components.
Cryo-FIB takes SEM to the next level. This technique is already popular in industry, particularly in the semiconductor industry. However, in the biological context, it has also shown value. Using a rapid frozen cryo sample on a cooled stage, the researcher can make depth profile images of a full thickness tissue sample.
Essentially SEM can be used to record an image and then a focussed beam of gallium or Xenon ions (1-2nm) can ablate or cut away controlled sections of tissue or cell to reveal the next level for imaging. In this way, for SEM, lipid and water containing structures are fixed by the freezing and can be observed in their natural state.
Site specific Cryo-FIB of a European ash leaf
LE BSE Detection
Precise sample preparation processes are essential for bio-samples. Some samples can suffer from charging effects, and can also be damaged by the high energy electron beam. Typically, biological samples have very fine surface features, and coating these specimens with various conductive materials can rapidly decrease the information from an acquired image.
Tissue and cells are often stained with an osmium fixative to enhance contrast. In these cases, use of a low energy back-scattered electron (BSE) detector is necessary for observing non-conductive substrate surrounding the prepared tissue or cell.
Low Energy BSE Detector Image of biological cells from thin section picked up on tape. Note the signal has been inverted so as to match typical TEM images
BSE are back-scattered out of the specimen by elastic scattering interactions with the specimen and is used to detect contrast between areas with different chemical compositions. With low energy BSE and its lower radiation damage, it is possible to observe the sample in its natural state without artefacts.
The LE BSE Detector allows BSE images to be acquired at accelerating voltages lower than can normally be acquired with a BSE detector. This means that large areas can be scanned and imaged more rapidly. This also allows BSE imaging of non-conductive samples at low accelerating voltages reducing charge build up and the size of the interaction volume and hence increasing the spatial resolution.
Tescan have advanced this area and the MIRA3 has the ability to rapidly scan and image a large area using LE-BSE but with high spatial resolution and 16k pixel imaging. The Tescan Image Snapper panorama software processing this data allows high pixel density panoramas to be acquired without downsizing providing researchers with a series of panoramic high-quality images of biological samples at high resolution.
MIRA3 is an SEM system with a fast image acquisition, an ultra-fast scanning system as well as dynamic and static compensation. The system has the capability for imaging at low and ultra-low electron landing energies using beam deceleration technology (BDT). BDT reduces optical aberration allowing focused spot sizes and high-resolution imaging at low energies. A variety of BSE detectors are available, including the LE-BSE detector.
The MIRA3 is also ideal for both low and high vacuum operation and has chamber geometries to fit all applications and can easily be configured with STEM detection to provide high spatial resolution imaging. The system can also be configured with a cryo-stage and cryo-transfer capability for low-temperature imaging for cryo-FIB and freeze-fracture experiments.
TESCAN USA Inc.
Founded in 1991 by a group of managers and engineers from Tesla with its electron microscopy history starting in the 1950’s, today TESCAN is a globally renowned supplier of Focused Ion Beam workstations, Scanning Electron Microscopes and Optical Microscopes. TESCAN’s innovative solutions and collaborative nature with its customers have won it a leading position in the world of nano- and microtechnology. The company is proud to participate in premier research projects with prominent institutions across a range of scientific fields. TESCAN provides its clients with leading-class products in terms of value, quality and reliability. TESCAN USA Inc. is the North American arm of TESCAN ORSAY HOLDINGS, a multinational company established by the merger of Czech company TESCAN, a leading global supplier of SEMs and Focused Ion Beam workstations, and the French company ORSAY PHYSICS, a world leader in customized Focused Ion Beam and Electron Beam technology.
This information has been sourced, reviewed and adapted from materials provided by TESCAN.
For more information on this source, please visit Tescan.com.