EM Techniques

The Otago Centre for Electon Microscopy and the Otago Centre for Confocal Microscopy together have a range of microscopes that cover a broad range of techniques. If you are unfamiliar with electron microscopy, read on for a description of the techniques that we are able to perform at the unit.

The Australian Microscopy and Microanalysis Research Facility (AMMRF) have produced an excellent interactive resource called My Scope to progress your training in advanced instrumentation and techniques. Modules are available for SEM, TEM, x-ray diffraction, AFM, confocal and microanalysis.

The Jensen Laboratory at Caltech have created a series of online YouTube tutorials covering cryo-TEM topics called Getting Started in Cryo-EM.



Scanning Electron Microscopy Techniques Available in the OCEM:

  • Secondary Electron Imaging
  • Backscattered Electron Imaging
  • Energy-Dispersive X-ray Spectroscopy
  • Electron Backscatter Diffraction Imaging
  • Cathodoluminescence
  • Variable Pressure SEM
  • High Resolution SEM
  • Cryo-SEM
  • Array Tomography


  • Transmission Electron Microscopy Techniques Available in the OCEM:

  • Conventional TEM
  • Cryoultramicrotomy
  • Immunocytochemistry
  • Correlative TEM
  • Freeze Fracture TEM
  • Metal Shadowing
  • Negative Staining
  • Heating and Cooling TEM
  • Single Particle Reconstruction
  • Cryo-TEM
  • Electron Tomography
  • Scanning Transmission Electron Microscopy (STEM)
  • High Pressure Freezing (HPF)


  • Scanning Electron Microscopy Techniques


    Scanning Electron Microscopy Techniques Available in the OCEM:


    Secondary Electron Imaging:

    Secondary Electron SEM

    Finely layered sediment from a 23 million year old volcanic lake, Middlemarch, Otago. Light-coloured summer layers of pennate diatoms are interbedded with a winter layer of plant debris, centric diatoms (yellow), freshwater sponges (green), and algal resting spores (red). Colours in this scanning electron microscope image are artificial. By: Uwe Kaulfuss.

    Provides high-resolution imaging of fine surface morphology.

    Secondary electron imaging can be performed on our Zeiss Sigma VP FEG SEM, JEOL FE-SEM6700, Cambridge 360 SEM (up to 150,000 sample dependent).





    Backscattered Electron Imaging:

    Back Scattered Electron

    Used to detect contrast between areas with different elemental compositions, as well as, surface topography. Higher atomic number material appears brighter than low atomic number material in a backscattered electron image. BSE images are very helpful for obtaining high-resolution compositional maps of a sample and for quickly distinguishing different phases. They are often used in conjunction with spot probe analyses by energy-dispersive x-ray spectroscopy.

    Backscattered electron imaging can be performed on our Zeiss Sigma VP FEG SEM, JEOL FE-SEM6700, Cambridge 360 SEM.

    Energy-Dispersive X-ray Spectroscopy (EDS or EDX):

    Energy-Dispersive X-Ray Spectroscopy

    An analytical method used for determining the elemental chemical composition of a sample, and to create element composition maps over a broader area. Element maps display element distributions in textural context, particularly for showing compositional zonation. EDS can either be used for qualitative or quantitative analysis. Qualitative analysis is used to determine which elements are present in a particular location. Quantitative analysis is used to find out the relative amounts of the elements, or how much of an element is present. Line profile analysis can also be performed with EDS.

    Energy-dispersive x-ray spectroscopy can be performed on our Zeiss Sigma VP FEG SEM, JEOL FE-SEM6700. Features or phases as small as a micron can be analyzed (sample dependent).

    Electron Backscatter Diffraction Imaging:

    Electron Backscatter Diffraction SEM

    Electron backscatter diffraction (EBSD) map of a sheared rock made mostly of grains of the mineral plagioclase. The red to green colour of each pixel shows the orientation. Yellow points are a different mineral (hornblende). Tone underlying colour shows changes in EBSD pattern quality. Blue lines are twin boundaries, where the crystal axes change by 180 degrees. By: Prof. David Prior.

    Used to determine crystal structures and orientations of minerals, which can be used to elucidate texture or preferred orientation of any crystalline or polycrystalline material. Applications include crystal orientation mapping, defect studies, phase identification, grain boundary and morphology studies, regional heterogeneity investigations, material discrimination, microstrain mapping, and physico-chemical identification (using complementary SEM techniques).

    Electron backscatter diffraction imaging can be performed on our Zeiss Sigma VP FEG SEM.

    Cathodoluminescence (CL):

    Provide information about the trace elements (typically transition metals or rare earth elements) contained in minerals and can be used to create compositional maps. Also provides information about processes such as crystal growth, replacement, deformation, provenance and the production of mechanically induced defects. Most commonly used in geological studies.

    Cathodoluminescence imaging is not available at present, but we hope to offer this service soon.

    Variable Pressure SEM (VP SEM):

    Conventional electron microscopes require that the electron-optic column and specimen chamber be under a high vacuum so that the electron beam can travel from the source to the sample without being scattered by residual gas atoms. This means that samples that are unable to withstand being under vacuum, or samples that would contaminate the vacuum system are unable to be imaged in conventional microscopes.

    Variable pressure scanning electron microscopes are able to operate with the specimen chamber at high and low pressures. Our new Zeiss Sigma VP FEG SEM is able to image specimens at pressures from 2 to 133 Pa. This allows samples that would otherwise be unsuitable for observation to be imaged. For example porous materials such as bone, wet or damp specimens, and biological specimens that cannot be maintained in their original state if they are allowed to dehydrate. The gaseous environment allows poorly conducting and insulating samples to be imaged without the need to coat them with a conducting metal layer. The gas in the specimen chamber also creates a microenvironment which can be used for a wide variety of in situ experiments (e.g. corrosion and oxidation).

    High Resolution SEM

    The secondary electrons generated as the electron beam enters the specimen (SE1), are localised within a few nanomenters of the impact sites of incident high-energy electrons and can respond to local fine-scale features. The SE1, in combination with BSE that retain a large fraction of the beam energy, is considered a 'high resolution' signal. High resolution SEM aims to either separate the high-resolution signals from the low-resolution signals, or use operating conditions that create similar ranges for all of the signals. We are able to perform high resolution SEM on our JEOL FE-SEM 6700 and Zeiss Sigma VP FEG SEM.

    Cryo-SEM

    Cryo-SEM

    Samples are solidified by plunge freezing in nitrogen and can withstand the vacuum conditions required for examination in the SEM while kept at low temperatures on the cryo-stage. This allows samples to be viewed in their hydrated state and is particularly useful for liquid, semi-liquid and beam sensitive samples. There is usually no exposure to toxic reagents and little mechanical damage to samples. Cryo-SEM is the most effective method of preventing sample water loss and retaining soluble materials, meaning there is less relocation of highly diffusable elements. Cryo-SEM can be used to study dynamic processes using a series of time resolved samples. Our JEOL FE-SEM 6700 has a cryo preparation stage and sample chamber.

    Array Tomography

    Resin-embedded tissue is serial sectioned onto a glass microscope slide. The sections are then stained with a fluorescent dye or tag and imaged with the confocal microscope. The sections can then be eluted and restained with another fluorescent tag or dye. After the desired labels have been imaged the sections are stained with a heavy metal and then imaged using backscatter electron microscopy. A three-dimensional model of the antigens, fluorescent proteins and ultrastructure in individual tissue specimens can then be constructed.



    Transmission Electron Microscopy Techniques


    Conventional TEM

    Conventional TEM

    Maturing sheep oocyte: I have been studying the ultrastructural differences between adult and prepubertal lamb oocytes to find reasons for the reduced ability of juvenile oocytes to produce viable embryos. This oocyte is surrounded by expanding cumulus cells and a zona pellucida layer. The cytoplasm is full of mitochondria, lipid droplets, transparent vesicles, and an MII nucleus. By: Karen Reader.

    A beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. The transmission of unscattered electrons is inversely proportional to the specimen thickness. Areas of the specimen that are more electron dense allow fewer transmitted unscattered electrons and appear darker, conversely the thinner areas permit more transmission and appear lighter. From this we can learn about the morphology of the specimen such as the size, shape and arrangement of the particles which make up the sample as well as their relationship to each other. TEM can also provide crystallographic information about the sample such as the arrangement of atoms in the specimen and their degree of order, detection of atomic-scale defects in areas a few nanometres in diameter. To prepare samples for TEM the specimens are typically fixed in glutaraldehyde, stained with uranyl acetate, embedded in resin and then cut into sections 70-80nm thin. Depending on the sample, the CM100 TEM is able to resolve images down to a few nanometres.

    Cryoultramicrotomy

    Rather than being dehydrated and embedded in resin, tissue is lightly fixed and then suspended in a matrix (gelatin). It is then infiltrated with a cryoprotectant (sucrose) and frozen in liquid nitrogen. Frozen sections are then cut from the block onto grids. This preparation method is particularly good for immunolabelling (see more information below).

    Immunocytochemistry

    Immunocytochemistry TEM

    Bundled GnRH neuron dendrites. The small black dots label the dendritic arms of two GnRH neurons (pseudocolored green and purple), cells in the brain that control fertility. The discovery that these two distinct dendritic elements, cut in the longitudinal plane, are found in close apposition and possess an attachment plate (shaded zone) between them, is suggestive of how these scattered cells communicate with one another. By: Dr. Rebecca Campbell.

    An antigen is localised by one or more antibodies that are applied to the cells. An electron-dense tag is attached directly or (more commonly) indirectly to the last of the added antibodies. Some tags are organic molecules that possess structured electron opacity (e.g. ferritin), others are enzymes whose reaction product can be detected after the addition of the substrate (e.g. horseradish peroxidase), and yet others are heavy metals that can visualised directly (e.g. colloidal gold).

    The only major requirement for localisation is that a specific antibody has been developed against the antigen. Other things to take into consideration are the size of the tag (whether it will infiltrate into the cellular structures), fixation and its effect on the antigenicity of the tissue, and whether to label before or after embedding. Pre-embedding labelling is best for surface labelling of live cells as antibodies do not penetrate cell membranes. Live cells may be broken open with detergents to reveal antigenic determinants (however ultrastructural preservation is compromised). It is also possible to use frozen ultrathin sections for immunocytochemistry. In the post-embedding technique cells are labelled on sections of tissue embedded in resin. Antigenicity may be compromised by fixation, but it is possible to etch tissue sections to expose antigenic sites.

    Correlative TEM

    Correlative Microscopy

    Breast cancer cells that have been immunolabelled for YB1 protein with a green fluorescent dye conjugated to gold particles (we used Life Technologies Alexa Fluor® 488 Goat Anti-Rabbit IgG, 5 nm Colloidal Gold Conjugate). The cell can be looked at an even higher magnification to see which cellular structures the 5nm gold has tagged to. By: Sharon Lequeux

    Correlative microscopy involves using multiple microscope systems to observe the same specimen, most commonly light and transmission electron microscopy. This technique pairs the high resolution of the electron microscope which is able to reveal the cellular ultrastructure with the ability to follow specific targets on or in living cells, revealing dynamic localisation and/or function of target molecules using fluorescence microscopy.

    Ultrathin sections of tissue are treated with a label that is able to be imaged under both microscopes, e.g. fluorescence photo-oxidation, quantum dots, double labelling with both fluorescent and gold conjugated antibodies, or fluoronanogold. The samples are first imaged using fluorescence microscopy and then transferred to the transmission electron microscope. Similar results can be achieved by taking parallel sections and preparing them either for light-microscopy or transmission electron microscopy.

    Other microscope systems can also be correlated, such as confocal and scanning electron microscopy (see array tomography), or scanning electron microscopy and transmission electron microscopy.

    Freeze Fracture TEM

    Freeze Fracture TEM

    The freeze-fracture technique consists of freezing and then physically breaking apart (fracturing) a biological sample and visualising the exposed structural detail of the fracture plane by vacuum-deposition of platinum-carbon to make a replica for examination in the transmission electron microscope. Freeze fracture can provide three-dimensional perspectives of cellular organization and details of membrane structure at macromolecular resolution. Of particular importance is the technique's ability to reveal the distribution and organization of integral membrane proteins as intramembrane particles in the membrane plane.

    Metal Shadowing

    Metal Shadowing

    Metal shadowing is a well established method for visualising small, isolated structures, such as macromolecules, in the TEM. The object here was to check a preparation for the correct configuration of myosin molecules. Replicas of purified myosin molecules were prepared for viewing by jet deposition on a molecularly-flat substrate. The molecules were then rotary shadowed at a low angle (6º) with platinum. By: Richard Easingwood and specimen provided by John Harris.

    A thin layer of evaporated metal, such as platinum, is laid at an angle on a biological sample. An acid bath dissolves the biological material, leaving a metal replica of its surface, which can then be examined in the transmission electron microscope. Variations in the angle and thickness of the deposited metal allow an image to be formed because some incident electrons will be scattered in various directions rather than pass through the preparation. If the metal is deposited mainly on one side of the sample, for instance, the image seems to have "shadows" where the metal appears dark and the shadows appear light. This techniques creates a three dimensional view of the specimen and is used to obtain information about the shapes of purified viruses, fibres, enzymes, and other subcellular particles.

    Negative Staining

    Negative Staining

    Bacteriophage isolated during a Microbiology undergraduate class. The students isolate and purify a bacteriophage and then characterize it. This includes preparing the specimen for imaging in the TEM. By: Jin Lee

    In negative staining the macromolecule itself is usually unstained but is instead surrounded by a dense stain. As a result, the specimen appears in negative contrast (lighter in tone against a dark background). Negative stains are not used on sectioned materials, but are used to contrast whole, intact biological structures (viruses, bacteria, cellular organelles) that have been deposited on a supporting plastic or carbon film.



    Heating and Cooling TEM

    Both a heat stage and a cool stage are available for use on the Philips CM100 and EM410 transmission electron microscopes. Please note that as the microscope operates under a high vacuum samples must be completely dry.



    Single Particle Reconstruction

    Single particle reconstruction takes many images of viruses or proteins at different orientations and averages them together to produce a 3D reconstruction of the particle. The combined image has much stronger and easily interpretable features than individual images which contain much more noise. The nominal resolution of the new microscope is 0.27 nanometres.



    Cryo-TEM

    Using the cryo-TEM samples can be viewed while frozen, avoiding chemical fixation and reducing damage to the samples caused by the electron beam.



    Electron Tomography

    Imaging of sections up to 300nm thick while tilting the specimen to 80°. The tilt series can then be used to create a 3D reconstruction of the sample's internal structures.



    Scanning Transmission Electron Microscopy (STEM)

    STEM (scanning transmission electron microscopy) enables the use of other of signals that cannot be spatially correlated in TEM, including secondary electrons, scattered beam electrons, characteristic X-rays, and electron energy loss - allowing very accurate elemental mapping and chemical analysis of samples.

  • High Pressure Freezing (HPF)