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Scanning electron microscopy reveals the form and ultrastructure of biological specimens
Scanning electron microscopy (SEM) offers a diverse range of techniques to study biological specimens at high magnification and resolution. By leveraging different imaging methods, researchers can gain valuable insights into the structure, morphology, and interactions of cells, tissues, and biomaterials.
Thermo Scientific SEMs deliver the techniques and applications required by life scientists and pharmaceutical researchers. Explore the diverse range of available SEM techniques and see how they can support your research; then, find which SEM best supports your needs.
Large-area 2D SEM mapping of biological samples
Two-dimensional SEM imaging allows for detailed visualization and analysis of surface topography and structure. Capture large (~100 mm) fields of view to understand composition and organization from surface topography down to subcellular resolutions. This method is particularly beneficial for the analysis of surfaces, cellular architecture, and the tissue organization of large-scale biological structures. Thermo Scientific Maps Software automates the acquisition of large, high-resolution sample overviews while also stitching the resulting tile set together. Maps Software can be used to seamlessly zoom between a bird’s eye overview and the desired final magnification, making it easy to search for rare events within the sample.
Gold-nanoparticle-labelled mouse cilia, imaged at multiple scales with the Thermo Scientific Apreo 2 SEM and Maps Software. A large area was captured at low resolution and then regions of interest were identified by clicking/zooming in on the overview; high-resolution images of these ROIs were then acquired. The multiscale workflow is seamlessly supported with Maps Software. Images courtesy of Nicolas Grillet, Stanford University.
Low-vacuum and environmental SEM for imaging of hydrated specimens
SEM is traditionally conducted under high-vacuum, offering excellent resolution and contrast with a wide range of detectors. This vacuum, however, can alter biological structures due to sample outgassing, meaning that these specimens require fixation and/or dehydration.
Low-vacuum (or variable pressure) SEM can accommodate partially hydrated and/or less conductive samples, making it a versatile choice for biological imaging. This mode allows for moderate water vapor pressure within the chamber, reducing the need for extensive sample preparation as well as potentially reducing charging artifacts during acquisition.
Environmental SEM (ESEM) takes low vacuum a step further by maintaining higher humidity levels in the chamber. This enables imaging of fully hydrated or even living specimens in near-native conditions. ESEM provides unique opportunities for real-time observation of dynamic processes and the imaging of biology at full turgor without the need for advanced sample preparation.
Papillae on the upper surface of a rose petal, captured in their natural state using environmental SEM. Courtesy of Marcos Rosado, Institut Catala de Nanociencia i Nanotecnologia.
Cryo SEM for high-resolution imaging of hydrated specimens
Working under cryogenic conditions provides an alternative to chemical fixation, as the rapid freezing of biological samples preserves their ultrastructure in a near-native state. This method allows the samples to remain fully hydrated, and imaging can be performed under high vacuum so that high-resolution surface topography can be acquired.
A cryo-stage in the SEM keeps the specimen frozen during imaging. Using this approach, samples can be examined in an unaltered (or minimally altered) state, providing biologically relevant insights. Aside from surface imaging, it is common to use an integrated, external preparation chamber to create fractures, conduct sublimations, and to coat samples with conductive metal layers.
Cryo-SEM image of a pollinated Ixeris chinensis flower at 500x magnification. Image courtesy of Dr. Wann-Neng Jane, Academia Sinica.
3D imaging of biological ultrastructure using volume electron microscopy
Biology happens in 3D, and many biological questions therefore require 3D insights, such as how proteins congregate in membranes, how organelles interact in cells, how cells arrange in tissues, how complex communities form within ecological niches, and more.
Volume electron microscopy (volume EM) is a group of techniques that provides 3D, multi-scale information on the ultrastructure of cells, tissues, and organisms at micrometer to nanometer resolution. Volume EM techniques are used across cell, tissue, and cancer biology, neuroscience, plant science, ecology, and biomaterials, enabling a deep understanding of form and function.
Within volume EM, several SEM-based techniques are used to generate ultrastructural reconstructions, including array tomography, serial block-face SEM (SBF-SEM) and focused ion beam SEM (FIB-SEM). These popular technique are adaptable and can be tailored to address specific questions.
Volume EM is especially valuable when it is necessary to know how something is distributed outside a single imaging plane, for instance, the way an axon runs through a brain sample or how the mitochondrial network flows through a cell. It can also help locate and capture rare events throughout a volume, such as synaptic vesicle fusion with a membrane, the site of a parasitic infection, or hyperplasic changes in a metastatic cancer nodules.
Cowpox-infected cell, imaged in 3D with the Thermo Scientific Volumescope 2 SEM. Thermo Scientific Amira Software was used to segment nuclei (green) as well as immature virions (orange) and mature viruses (yellow). Sample courtesy of Michael Laue, Robert Koch Institute, Berlin, Germany.
Correlative microscopy
Correlative light and electron microscopy (CLEM) combines fluorescence microscopy with high-resolution SEM imaging, enabling researchers to bridge the gap between molecular localization and ultrastructural details. Beyond fluorescence, correlative imaging can integrate data from various other techniques, such as X-ray computed tomography (CT) as well as elemental and vibrational spectroscopy. X-ray CT provides a lower resolution 3D overview of the whole specimen, which can be used to identify regions of interest for higher resolution acquisition with SEM. Elemental analysis, meanwhile, provides additional compositional information that can be mapped onto the SEM images, generating a more thorough understanding of the sample as a whole. Collectively, such approaches can provide comprehensive insights into a sample’s morphology, composition, and function. Correlative imaging with Thermo Scientific SEMs is powered by Maps Software, which provides accurate and efficient data collection, correlation, and visualization.
Mouse brain imaged with widefield fluorescence microscopy and SEM. Fluorescence data shows myelin basic protein (red), GABA (green), glutamine synthase (blue), and DAPI (teal), overlaid on the greyscale SEM image (obtained with the Apreo 2 SEM). Correlative overlay of the immuno-fluorescence and SEM data was performed with Maps Software. Sample and light microscopy images courtesy of Kristina Micheva, Stanford University.
Correlation of upstream fluorescence microscopy data with SEM, FIB-SEM, and TEM in Maps Software.
Scanning transmission electron microscopy in SEM
A scanning transmission electron microscopy (STEM) mode in SEM enables imaging of ultra-thin resin-embedded biological sections, resulting in TEM-like images. The introduction of a STEM detector to an SEM can provide high-resolution analysis of soft tissue sections at low energy, providing high contrast while preserving the structure of the sample. This offers improved contrast compared to traditional SEM backscattered electron imaging, which may be preferable for minimally stained or high-magnification studies.
Murine kidney imaged with an Apreo 2 SEM using a scanning transmission electron microscopy detector. Maps Software was used to acquire the larger area followed by targeted acquisition of the mitochondria at higher resolution.
Section of a planarian worm, imaged on a Thermo Scientific Verios SEM in STEM mode. Maps Software automates the acquisition of large overviews at high resolution and is also used to stitch the resulting tile set. Users can seamlessly zoom between an overview and the native image resolution. Sample courtesy of Melainia McClain, Stowers Institute for Medical Research.
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