An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.
The electron is a subatomic particle, symbol
, whose electric charge is negative one elementary charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary particles because they have no known components or substructure. The electron has a mass that is approximately 1/1836 that of the proton. Quantum mechanical properties of the electron include an intrinsic angular momentum (spin) of a half-integer value, expressed in units of the reduced Planck constant, ħ. As it is a fermion, no two electrons can occupy the same quantum state, in accordance with the Pauli exclusion principle. Like all elementary particles, electrons exhibit properties of both particles and waves: they can collide with other particles and can be diffracted like light. The wave properties of electrons are easier to observe with experiments than those of other particles like neutrons and protons because electrons have a lower mass and hence a longer de Broglie wavelength for a given energy.
The photon is a type of elementary particle, the quantum of the electromagnetic field including electromagnetic radiation such as light, and the force carrier for the electromagnetic force. The photon has zero rest mass and always moves at the speed of light within a vacuum.
Angular resolution or spatial resolution describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object, thereby making it a major determinant of image resolution. In physics and geosciences, the term spatial resolution refers to the precision of a measurement with respect to space.
Electron microscopes have electron optical lens systems that are analogous to the glass lenses of an optical light microscope.
Electron optics is a mathematical framework for the calculation of electron trajectories along electromagnetic fields. The term optics is used because magnetic and electrostatic lenses act upon a charged particle beam similarly to optical lenses upon a light beam.
Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the images.
Ultrastructure is the architecture of cells and biomaterials that is visible at higher magnifications than found on a standard optical light microscope. This traditionally meant the resolution and magnification range of a conventional transmission electron microscope (TEM) when viewing biological specimens such as cells, tissue, or organs. Ultrastructure can also be viewed with scanning electron microscopy and super-resolution microscopy, although TEM is a standard histology technique for viewing ultrastructure. Such cellular structures as organelles, which allow the cell to function properly within its specified environment, can be examined at the ultrastructural level.
The cell is the basic structural, functional, and biological unit of all known living organisms. A cell is the smallest unit of life. Cells are often called the "building blocks of life". The study of cells is called cell biology.
A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist involving extraction of sample cells or tissues for examination to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically. When an entire lump or suspicious area is removed, the procedure is called an excisional biopsy. An incisional biopsy or core biopsy samples a portion of the abnormal tissue without attempting to remove the entire lesion or tumor. When a sample of tissue or fluid is removed with a needle in such a way that cells are removed without preserving the histological architecture of the tissue cells, the procedure is called a needle aspiration biopsy. Biopsies are most commonly performed for insight into possible cancerous and inflammatory conditions.
In 1926 Hans Busch developed the electromagnetic lens.
According to Dennis Gabor, the physicist Leó Szilárd tried in 1928 to convince him to build an electron microscope, for which he had filed a patent.The first prototype electron microscope, capable of four-hundred-power magnification, was developed in 1931 by the physicist Ernst Ruska and the electrical engineer Max Knoll. The apparatus was the first practical demonstration of the principles of electron microscopy. In May of the same year, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained a patent for an electron microscope. In 1932, Ernst Lubcke of Siemens & Halske built and obtained images from a prototype electron microscope, applying the concepts described in Rudenberg's patent.
Dennis Gabor ; Hungarian: Gábor Dénes; 5 June 1900 – 9 February 1979) was a Hungarian-British electrical engineer and physicist, most notable for inventing holography, for which he later received the 1971 Nobel Prize in Physics.
Ernst August Friedrich Ruska was a German physicist who won the Nobel Prize in Physics in 1986 for his work in electron optics, including the design of the first electron microscope.
Max Knoll was a German electrical engineer.
In the following year, 1933, Ruska built the first electron microscope that exceeded the resolution attainable with an optical (light) microscope. [ clarification needed ] Although current transmission electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype.[ citation needed ]Four years later, in 1937, Siemens financed the work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska, Ernst's brother, to develop applications for the microscope, especially with biological specimens. Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope. Siemens produced the first commercial electron microscope in 1938. The first North American electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus. Siemens produced a transmission electron microscope (TEM) in 1939.
Bodo von Borries was a German physicist. He was the co-inventor of the electron microscope.
Helmut Ruska was a German physician and biologist from Heidelberg. After earning his medical degree, he spent several years working as a physician at hospitals in Heidelberg and Berlin. During this time, he also worked closely with his brother Ernst Ruska (1906-1988) and brother-in-law Bodo von Borries (1905-56), who were both research scientists at Siemens-Reiniger-Werke. Ernst Ruska was the inventor of the electron microscope, and later winner of a Nobel Prize. From 1948-1951, Helmut Ruska was a professor at the University of Berlin, in 1952 he moved to the United States where he was a micromorphologist for the New York State Department of Health in Albany. He returned to Germany in 1958 as director of biophysics at the University of Düsseldorf.
Manfred von Ardenne was a German research and applied physicist and inventor. He took out approximately 600 patents in fields including electron microscopy, medical technology, nuclear technology, plasma physics, and radio and television technology. From 1928 to 1945, he directed his private research laboratory Forschungslaboratorium für Elektronenphysik. For ten years after World War II, he worked in the Soviet Union on their atomic bomb project and was awarded a Stalin Prize. Upon his return to the then East Germany, he started another private laboratory, Forschungsinstitut Manfred von Ardenne.
The original form of the electron microscope, the transmission electron microscope (TEM), uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. The image detected by the digital camera may be displayed on a monitor or computer.
The resolution of TEMs is limited primarily by spherical aberration, but a new generation of hardware correctors can reduce spherical aberration to increase the resolution in high-resolution transmission electron microscopy (HRTEM) to below 0.5 angstrom (50 picometres),enabling magnifications above 50 million times. The ability of HRTEM to determine the positions of atoms within materials is useful for nano-technologies research and development.
Transmission electron microscopes are often used in electron diffraction mode. The advantages of electron diffraction over X-ray crystallography are that the specimen need not be a single crystal or even a polycrystalline powder, and also that the Fourier transform reconstruction of the object's magnified structure occurs physically and thus avoids the need for solving the phase problem faced by the X-ray crystallographers after obtaining their X-ray diffraction patterns of a single crystal or polycrystalline powder.
One major disadvantage of the transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Creating these thin sections for biological and materials specimens is technically very challenging. Semiconductor thin sections can be made using a focused ion beam. Biological tissue specimens are chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers, and similar materials may require staining with heavy atom labels in order to achieve the required image contrast.
One application of TEM is serial-section electron microscopy (ssEM), for example in analyzing the connectivity in volumetric samples of brain tissue by imaging many thin sections in sequence.
The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). When the electron beam interacts with the specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission, all of which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. The image displayed by an SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown below and to the right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs.
Generally, the image resolution of an SEM is lower than that of a TEM. However, because the SEM images the surface of a sample rather than its interior, the electrons do not have to travel through the sample. This reduces the need for extensive sample preparation to thin the specimen to electron transparency. The SEM is able to image bulk samples that can fit on its stage and still be maneuvered, including a height less than the working distance being used, often 4 millimeters for high-resolution images. The SEM also has a great depth of field, and so can produce images that are good representations of the three-dimensional surface shape of the sample. Another advantage of SEMs comes with environmental scanning electron microscopes (ESEM) that can produce images of good quality and resolution with hydrated samples or in low, rather than high, vacuum or under chamber gases. This facilitates imaging unfixed biological samples that are unstable in the high vacuum of conventional electron microscopes.
In their most common configurations, electron microscopes produce images with a single brightness value per pixel, with the results usually rendered in grayscale.However, often these images are then colorized through the use of feature-detection software, or simply by hand-editing using a graphics editor. This may be done to clarify structure or for aesthetic effect and generally does not add new information about the specimen.
In some configurations information about several specimen properties is gathered per pixel, usually by the use of multiple detectors.In SEM, the attributes of topography and material contrast can be obtained by a pair of backscattered electron detectors and such attributes can be superimposed in a single color image by assigning a different primary color to each attribute. Similarly, a combination of backscattered and secondary electron signals can be assigned to different colors and superimposed on a single color micrograph displaying simultaneously the properties of the specimen.
Some types of detectors used in SEM have analytical capabilities, and can provide several items of data at each pixel. Examples are the Energy-dispersive X-ray spectroscopy (EDS) detectors used in elemental analysis and Cathodoluminescence microscope (CL) systems that analyse the intensity and spectrum of electron-induced luminescence in (for example) geological specimens. In SEM systems using these detectors, it is common to color code the signals and superimpose them in a single color image, so that differences in the distribution of the various components of the specimen can be seen clearly and compared. Optionally, the standard secondary electron image can be merged with the one or more compositional channels, so that the specimen's structure and composition can be compared. Such images can be made while maintaining the full integrity of the original signal, which is not modified in any way.
In the reflection electron microscope (REM) as in the TEM, an electron beam is incident on a surface but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is detected. This technique is typically coupled with reflection high energy electron diffraction (RHEED) and reflection high-energy loss spectroscopy (RHELS).[ citation needed ] Another variation is spin-polarized low-energy electron microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains.
The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion. Often TEM can be equipped with the scanning option and then it can function both as TEM and STEM.
Materials to be viewed under an electron microscope may require processing to produce a suitable sample. The technique required varies depending on the specimen and the analysis required:
Electron microscopes are expensive to build and maintain, but the capital and running costs of confocal light microscope systems now overlaps with those of basic electron microscopes. Microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling systems.
The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. An exception is liquid-phase electron microscopy 20 Torr or 2.7 kPa) wet environment. Various techniques for in situ electron microscopy of gaseous samples have been developed as well.using either a closed liquid cell or an environmental chamber, for example, in the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to
Scanning electron microscopes operating in conventional high-vacuum mode usually image conductive specimens; therefore non-conductive materials require conductive coating (gold/palladium alloy, carbon, osmium, etc.). The low-voltage mode of modern microscopes makes possible the observation of non-conductive specimens without coating. Non-conductive materials can be imaged also by a variable pressure (or environmental) scanning electron microscope.
Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for example) require no special treatment before being examined in the electron microscope. Samples of hydrated materials, including almost all biological specimens have to be prepared in various ways to stabilize them, reduce their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may result in artifacts , but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods. Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.
Biology and life sciences
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
A microscope is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope.
A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the detected signal to produce an image. SEM can achieve resolution better than 1 nanometer. Specimens are observed in high vacuum in conventional SEM, or in low vacuum or wet conditions in variable pressure or environmental SEM, and at a wide range of cryogenic or elevated temperatures with specialized instruments.
Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a charge-coupled device.
Photoemission electron microscopy is a type of electron microscopy that utilizes local variations in electron emission to generate image contrast. The excitation is usually produced by ultraviolet light, synchrotron radiation or X-ray sources. PEEM measures the coefficient indirectly by collecting the emitted secondary electrons generated in the electron cascade that follows the creation of the primary core hole in the absorption process. PEEM is a surface sensitive technique because the emitted electrons originate from a shallow layer. In physics, this technique is referred to as PEEM, which goes together naturally with low-energy electron diffraction (LEED), and low-energy electron microscopy (LEEM). In biology, it is called photoelectron microscopy (PEM), which fits with photoelectron spectroscopy (PES), transmission electron microscopy (TEM), and scanning electron microscopy (SEM).
A scanning transmission electron microscope (STEM) is a type of transmission electron microscope (TEM). Pronunciation is [stɛm] or [ɛsti:i:ɛm]. As with a conventional transmission electron microscope (CTEM), images are formed by electrons passing through a sufficiently thin specimen. However, unlike CTEM, in STEM the electron beam is focused to a fine spot which is then scanned over the sample in a raster illumination system constructed in a way that at each point sample illuminated with the beam parallel to the optical axis. The rastering of the beam across the sample makes STEM suitable for analytical techniques such as Z-contrast annular dark-field imaging, and spectroscopic mapping by energy dispersive X-ray (EDX) spectroscopy, or electron energy loss spectroscopy (EELS). These signals can be obtained simultaneously, allowing direct correlation of images and spectroscopic data.
Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of cryogenic electron microscopy, more specifically a type of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures. Cryo-EM is gaining popularity in structural biology.
Focused ion beam, also known as FIB, is a technique used particularly in the semiconductor industry, materials science and increasingly in the biological field for site-specific analysis, deposition, and ablation of materials. A FIB setup is a scientific instrument that resembles a scanning electron microscope (SEM). However, while the SEM uses a focused beam of electrons to image the sample in the chamber, a FIB setup uses a focused beam of ions instead. FIB can also be incorporated in a system with both electron and ion beam columns, allowing the same feature to be investigated using either of the beams. FIB should not be confused with using a beam of focused ions for direct write lithography. These are generally quite different systems where the material is modified by other mechanisms.
Ultramicrotomy is a method for cutting specimens into extremely thin slices, called ultra-thin sections, that can be studied and documented at different magnifications in a transmission electron microscope (TEM). It is used mostly for biological specimens, but sections of plastics and soft metals can also be prepared. Sections must be very thin because the 50 to 125 kV electrons of the standard electron microscope cannot pass through biological material much thicker than 150 nm. For best resolutions, sections should be from 30 to 60 nm. This is roughly the equivalent to splitting a 0.1 mm-thick human hair into 2,000 slices along its diameter, or cutting a single red blood cell into 100 slices.
Electron cryotomography (CryoET) is an imaging technique used to produce high-resolution (~1-4 nm) three-dimensional views of samples, typically biological macromolecules and cells. CryoET is a specialized application of transmission electron cryomicroscopy (CryoTEM) in which samples are imaged as they are tilted, resulting in a series of 2D images that can be combined to produce a 3D reconstruction, similar to a CT scan of the human body. In contrast to other electron tomography techniques, samples are immobilized in non-crystalline ("vitreous") ice and imaged under cryogenic conditions, allowing them to be imaged without dehydration or chemical fixation, which could otherwise disrupt or distort biological structures.
Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures of sub-cellular macro-molecular objects. Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data. In the process, a beam of electrons is passed through the sample at incremental degrees of rotation around the center of the target sample. This information is collected and used to assemble a three-dimensional image of the target. For biological applications, the typical resolution of ET systems are in the 5–20 nm range, suitable for examining supra-molecular multi-protein structures, although not the secondary and tertiary structure of an individual protein or polypeptide.
Electron-beam-induced deposition (EBID) is a process of decomposing gaseous molecules by an electron beam leading to deposition of non-volatile fragments onto a nearby substrate. The electron beam is usually provided by a scanning electron microscope, which results in high spatial accuracy and the possibility to produce free-standing, three-dimensional structures.
The environmental scanning electron microscope or ESEM is a scanning electron microscope (SEM) that allows for the option of collecting electron micrographs of specimens that are "wet," uncoated, or both by allowing for a gaseous environment in the specimen chamber.
Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.
Scanning confocal electron microscopy (SCEM) is an electron microscopy technique analogous to scanning confocal optical microscopy (SCOM). In this technique, the studied sample is illuminated by a focussed electron beam, as in other scanning microscopy techniques, such as scanning transmission electron microscopy or scanning electron microscopy. However, in SCEM, the collection optics is arranged symmetrically to the illumination optics to gather only the electrons that pass the beam focus. This results in superior depth resolution of the imaging. The technique is relatively new and is being actively developed.
Low-voltage electron microscope (LVEM) is an electron microscope which operates at accelerating voltages of a few kiloelectronvolts or less. Traditional electron microscopes use accelerating voltages in the range of 10-1000 keV.
Serial block-face scanning electron microscopy is a method to generate high resolution three-dimensional images from small samples. The technique was developed for brain tissue, but it is widely applicable for any biological samples. A serial block-face scanning electron microscope consists of an ultramicrotome mounted inside the vacuum chamber of a scanning electron microscope. Samples are prepared by methods similar to that in transmission electron microscopy (TEM), typically by fixing the sample with aldehyde, staining with heavy metals such as osmium and uranium then embedding in an epoxy resin. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. This sequence of sample imaging, section cutting and block raising can acquire many thousands of images in perfect alignment in an automated fashion. Practical SBFSEM was invented in 2004 by Winfried Denk at the Max-Planck-Institute in Heidelberg and is commercially available from Gatan Inc. and Thermo Fisher Scientific (VolumeScope).
In situ electron microscopy is an investigatory technique where an electron microscope is used to watch a sample's response to a stimulus in real time. Due to the nature of the high-energy beam of electrons used to image a sample in an electron microscope, microscopists have long observed that specimens are routinely changed or damaged by the electron beam. Starting in the 1960s, and using Transmission Electron Microscopes (TEMs), scientists made deliberate attempts to modify materials while the sample was in the specimen chamber, and to capture images through time of the induced damages.
Scanning transmission X-ray microscopy (STXM) is a type of X-ray microscopy in which a zone plate focuses an X-ray beam onto a small spot, a sample is scanned in the focal plane of the zone plate and the transmitted X-ray intensity is recorded as a function of the sample position. A stroboscopic scheme is used where the excitation is the pump and the synchrotron X-ray flashes are the probe. X-ray microscopes work by exposing a film or charged coupled device detector to detect X-rays that pass through the specimen. The image formed is of a thin section of specimen. Newer X-ray microscopes use X-ray absorption spectroscopy to heterogeneous materials at high spatial resolution. The essence of the technique is a combination of spectromicroscopy, imaging with spectral sensitivity, and microspectroscopy, recording spectra from very small spots.
Liquid-phase electron microscopy refers to a class of methods for imaging specimens in liquid with nanometer spatial resolution using electron microscopy. LP-EM overcomes the key limitation of electron microscopy: since the electron optics requires a high vacuum, the sample must be stable in a vacuum environment. Many types of specimens relevant to biology, materials science, chemistry, geology, and physics, however, change their properties when placed in a vacuum.
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