Electron Microscopy Facility - FAQ's
Frequently Asked Questions
What information does the TEM provide? Why would I use it?
- The transmission electron microscope (TEM) is used to examine the structure, composition, and properties of specimens in submicron detail. It can be used for:
- Image morphology of samples, e.g. view sections of material, fine powders suspended on a thin film, small whole organisms such as viruses or bacteria, and frozen solutions.
- Tilt a sample and collect a series of images to construct a 3-dimensional image.
- View frozen material (in a TEM with a cryostage). There are some things TEM can't do:
- TEM cannot take colour images. Colour is sometimes added artificially to TEM images.
- TEM cannot image through thick samples: the usual sample thickness is around 100-200nm. Electrons cannot readily penetrate sections much thicker than 200nm.
- The TEM cannot reliably image charged molecules that are mobile in a matrix. For example, some species (e.g. Na+) are volatile under the electron beam because the negative electron beam exerts a force on charged material.
How long does it take to prepare typical samples for viewing in the electron microscope?
- For the TEM, upon receiving a live sample, a typical turn-around time is ~1-2 weeks for fixation, embedding, sectioning and contrast staining grids before they can be imaged.
Why does the sample size have to be so small?
- For the TEM, samples must ideally be less than 1 mm3 to begin with for good preservation of morphology during the fixation stage. The main limitation is the speed and extent of penetration of fixatives (typically glutaraldehyde). If the fixative doesn't penetrate throughout the tissue, then it will be poorly preserved. So if, for example, we want to look at a insect wing, the wing will have to initially be cut into lots of tiny pieces, each around <1 mm3, at the time of fixation. Then, in general, we will only section one or two small pieces to get an idea of what the sample is like. Remember, if you’re going to have to cut slices of the sample that are only ~100 nm thick for imaging, then 1 mm provides ~10,000 slices — way too many to search for a rare feature. To "preview" or find interesting sections of the sample, we typically perform light microscopy of stained "semi-thin" sections (typically 0.5 µm) first. We would then focus our efforts in thinner slices.
Why can’t we image biological samples in native state?
- Biological samples are problematic to image with electrons for several key reasons. The requirement for a vacuum.
Requirement for vacuum
- Biological material is not naturally resistant to vacuums. The high water content and water permeable membranes/specimen surfaces result in a rapid evaporation of water and subsequent distortion of biological tissue. Dehydration of the sample with solvents typically results in a coagulation of cellular components. This changes the ultrastructure of the sample. Fixation via freezing or chemicals can reduce dehydration artifacts and, combined with the removal of water, prevent sample distortion in the vacuum.
- The sample thickness is very important in TEM. Thickness of the sample limits the amount of resolution that can be obtained in an image. The ratio is usually resolution = 1/10 sample thickness. Two thick and the electrons will not penetrate the sample. The sample thickness necessary is often much smaller than the thickness of a cell. Slicing biological tissue that thin is problematic as the tissue usually collapses. Solutions involve freezing the tissue and cutting it while it is frozen (cryo-ultramicrotomy), or embedding the sample in plastic resins, which provides enough support to permit thin sections of tissue to be cut (ultramicrotomy). Typically the sections are 50-70 nm thick. Thickness is not usually a problem for imaging proteins and viruses, which can be placed on the supporting grid whole.
- Biological tissue is known as a weak phase sample. The tissue has very low electron density, which results in few electron scattering events and low contrast images. Frozen samples that have no stain can be imaged as there is enough density difference between the sample and surrounding frozen water. For resin embedded samples the density difference is much lower and electron dense stains are necessary, often applied before and after resin embedding.
- Biological tissue is, generally, non-conductive. Electrons carry a negative charge and bombarding biological samples with negative charge can cause a charge build up. In both the SEM and TEM a charging sample can result in image distortion. In the TEM this is most often observed as "drift", which creates a blur in the image.he requirement for a vacuum.
What is scanning electron microscopy (SEM)?
- A scanning electron microscope (SEM) produces surface images by scanning a specimen with a beam of high energy electrons in a raster pattern. The electrons interact with the atoms that make up the specimen, producing signals that contain information about the specimen's surface, topography, composition and other properties.
- Depending on the instrument, resolution will vary between less than 1nm and 20nm. In addition to its high resolving capability, the SEM also has a great depth of field, giving the characteristic three-dimensional appearance that is useful for understanding the surface structure of a specimen.
- Many SEMs also have a facility to analyse the X-rays given off by the specimen as a result of its electron bombardment. As each element in the periodic table produces its own X-ray spectrum, this can be used to identify the elemental composition and measure the abundance of elements in the specimen.
What is scanning electron microscopy (SEM) specimen preparation?
- Preparation instruments such as critical point dryers and freeze dryers are designed to controllably remove water from biological and similar liquid-based specimens. This partially overcomes the adverse effects of air drying (namely, severe collapse of cellular structure as the drying front passes through the specimen).
- Prior to drying, most biological specimens require chemical stabilization in order to preserve them and allow them to better withstand subsequent drying and sputter coating processes.
- Many modern SEMs can operate without exposing the specimen to high vacuum. Often referred to as environmental/low-vacuum/high-pressure/variable-pressure SEMs, these instruments use higher pressures to minimise out-gassing from volatile specimens. However, variable pressure techniques are generally limited to certain specimen types and have a number of disadvantages in terms of specimen stability and the information that can be obtained.
- For water-based, liquid or semi-liquid and beam-sensitive materials, cryo-SEM overcomes many of the problems associated with drying protocols and variable pressure techniques.
- For most protocols, SEM specimens need to be electrically conductive. For this reason, non-conductive and semi-conductive SEM specimens require the deposition of a thin surface layer of metal. This also increases the amount of secondary electrons that can be detected from the surface of the specimen in the SEM, and therefore increases the signal to noise ratio.
- Metal layers need to have a fine grain and be evenly distributed across the specimen surface. We offer a wide choice of sputter coaters, carbon coaters and vacuum evaporators designed to suit most specimen types - see Product Information.
What is cryo-SEM?
- Cryo preparation techniques for scanning electron microscopy (cryo-SEM) are now considered essential for the successful observation of many wet or ‘beam sensitive’ specimens.
- Cryo-SEM of biological material removes the need for conventional preparation methods, such as chemical fixation and critical point drying, and allows observation of the specimen in its ‘natural’ hydrated state.
Why choose cryo-SEM?
The limitations of conventional ‘wet processing’ include:
* Shrinkage and distortion
* Extraction of soluble materials
* Mechanical damage (fragile specimens can be damaged during conventional processing)
* Slow (24 hours or longer)
* Toxic reagents are required (fixatives, buffers etc)
Advantages of cryo-SEM:
* Specimen viewed in fully hydrated state
* Soluble materials are retained
* Little or no mechanical damage
* Time lapse experiments and evaluating industrial processes at timed intervals
* Usually no exposure to toxic reagents
* Rapid process
* High resolution capability (compared to low-vacuum techniques)
* Extra information obtained by low-temperature fracturing (compared with conventional and low-vacuum methods)
* Good for liquid, semi-liquids and beam sensitive specimens
* Ability to selectively etch (sublimate to reveal information)
* Ability to ‘rework’ specimen (eg re-fracture and coat)