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Industrial Liaison Group:
Tel: +44 (0) 1235 778797
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It's day 9 of the Diamond Industrial Liaison advent calendar. Today is all about electron microscopy for biological sciences. We introduce you to our new facility eBIC - electron Biological Imaging Centre and give you a small overview the amazing discoveries made by this incredible technique
Most people will have used a microscope at some point in their life, perhaps in school to observe the wings of an insect or to look at a sample of blood cells. These types of microscopes illuminate the surface of your subject of study and use transparent glass lenses to magnify the image of whatever you are observing. However, the size of the smallest features that can be distinguished under the microscope is on the order of the wavelength of the light used. Visible light, which is the one our eyes are sensitive to, ranges between 390 and 700 nanometres (one nanometre is one billionth of a metre). This means that we cannot observe things that are smaller than a few hundred nanometres using our eyes and visible light.
With the advancement of science and technology, there is a whole world of research that can be carried out on samples at small scales, at just fractions of a nanometre. An electron microscope allows us to see at these small scales.
Electron microscopes are a type of microscope that uses a beam of electrons to create an image of the specimen. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail. They are large, expensive pieces of equipment, but their immense power has led to great advances in the scientific research world and in particular in the area of biological sciences
There are two types of electron microscopy; Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy.
Transmission electron microscopy (TEM)
This type of microscopy involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the structure of the specimen. The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer.
Transmission electron microscopes produce two-dimensional, black and white images. Resolution of the TEM can be limited by spherical and chromatic aberration (an optical effect occurring when the oblique rays entering a lens are focused in a different location than the central rays), but a new generation of aberration correctors has been create that are able to overcome or limit these aberrations. These advances have allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.089 nm and atoms in silicon at 0.078 nm at magnifications of 50 million times. It is this ability to determine the positions of atoms within materials which has made the TEM an indispensable tool for nano-technologies research and development in many fields, including heterogeneous catalysis and the development of semiconductor devices for electronics and photonics.
TEMs are typically used for viewing internal features that are inside or beyond the surface (e.g. organelles, macromolecules, atoms). It is also possible for TEMs to be capable of 3-D tomography which involves taking a succession of images whilst tilting the specimens through increasing angles, which can then be combined to form a three-dimensional image of the specimen.
Scanning Electron Microscope (SEM)
Unlike the TEM, at no time does a Scanning Electron Microscope (SEM) carry a complete image of the specimen. Whereas in TEM the electrons in the primary beam are transmitted through the sample, SEM produces images by detecting secondary electrons that are emitted from the surface due to excitation from a primary electron beam. In more detail, SEM works by rapidly scanning your sample with a focused electron beam. This causes electrons to be knocked off the surface of your sample. These secondary electrons provide signals carrying information about the properties of the specimen surface, such as its topography and composition. And it is these secondary or backscattered electrons that are picked up by a detector and are used to produce your image.
Because of the way it works, SEM focuses on the sample’s surface and its composition and shows the sample bit by bit through scanning. Things you can see with a SEM include the surface of a housefly’s eye, human inner ear hair cells, or elements on the head of a pin. Similar to the limitations of TEMs, SEMs yield only greyscale images and it is not possible to observe living specimens as the entire system must be in a vacuum in order for the image to be formed. However, on the plus side, since the SEM image relies on electron interactions at the surface rather than transmission, it is able to image thicker samples and has a much greater depth of view. Hence, SEM images are three-dimensional giving more accurate representations than TEMs.
TEM and SEM do share a number of similarities:
Cryo-electron microscopy
Cryo-electron microscopy is a method for imaging frozen-hydrated specimens at cryogenic temperatures (-180°C for liquid nitrogen stages, -269°C for He). A tiny drop of the sample is placed onto a copper grid, the sample is 'blotted' to leave a thin layer of molecules and then plunged into something like liquid ethane to freeze it so fast that ice crystals can't form. The grid containing the the frozen molecules is then loaded into the electron microscope and the molecules, when shot with the electron beam, leave a unique "shadow" on the detector. These "shadows" contain all the 3-dimensional information of the molecule, compressed into a 2D image. By combining all the images of molecules in all their various orientations, a 3D structure can be created.
In cryo-electron microscopy, specimens remain in their native state without the need for dyes or fixatives, allowing the study of fine cellular structures, viruses and protein complexes at molecular resolution.
We’re always happy to discuss any enquiries or talk about ways in which access to Diamond’s facilities may be beneficial to your business so please do give us a call on 01235 778797 or send us an e-mail. You can keep in touch with the latest development by following us on Twitter @DiamondILO orLinkedIn.
Diamond Light Source is the UK's national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire.
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