Liquid-Phase Electron Microscopy

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TEM of a specimen in liquid enclosed by two membrane windows supported by silicon microchips. The thickness of the liquid t is kept sufficiently small with respect to the mean free path length of electron scattering in the materials, so that the electron beam is transmitted through the sample for detection. The membrane windows bulge outward into the vacuum. Lctem3.svg
TEM of a specimen in liquid enclosed by two membrane windows supported by silicon microchips. The thickness of the liquid t is kept sufficiently small with respect to the mean free path length of electron scattering in the materials, so that the electron beam is transmitted through the sample for detection. The membrane windows bulge outward into the vacuum.
ESEM of nanoparticles in liquid placed in a vacuum chamber containing a background pressure of vapor. The sample support stage is cooled to achieve condensation, for example, to 4 degC for 813 Pa water vapor. The electron optics in high vacuum is separated from the sample chamber by a pump limiting aperture. Detection of backscattered or secondary electrons is optimal when applying a positive electrical potential V between the sample and the detector, so that a cascade of electrons and ions is created. Esem2.svg
ESEM of nanoparticles in liquid placed in a vacuum chamber containing a background pressure of vapor. The sample support stage is cooled to achieve condensation, for example, to 4 °C for 813 Pa water vapor. The electron optics in high vacuum is separated from the sample chamber by a pump limiting aperture. Detection of backscattered or secondary electrons is optimal when applying a positive electrical potential V between the sample and the detector, so that a cascade of electrons and ions is created.

Liquid-phase electron microscopy (LP EM) 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.

The ability to study liquid samples, particularly those involving water, with electron microscopy has been a wish ever since the early days of electron microscopy [1] but technical difficulties prevented early attempts from achieving high resolution. [2] Two basic approaches exist for imaging liquid specimens: i) closed systems, mostly referred to as liquid cell EM (LC EM), and ii) open systems, often referred to as environmental systems. In closed systems, thin windows made of materials such as silicon nitride or graphene are used to enclose a liquid for placement in the microscope vacuum. Closed cells have found widespread use in the past decade due to the availability of reliable window microfabrication technology. [3] [4] Graphene provides the thinnest possible window. [5] The oldest open system that gained widespread usage was environmental scanning electron microscopy (ESEM) of liquid samples on a cooled stage in a vacuum chamber containing a background pressure of vapor. [6] [7] Low vapor pressure liquids such as ionic liquids can also be studied in open systems. [8] LP-EM systems of both open and closed type have been developed for all three main types of electron microscopy, i.e., transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and scanning electron microscope (SEM). [9] Instruments integrating liquid-phase SEM with light microscopy have also been developed. [10] [11] Electron microscopic observation in liquid has been combined with other analytical methods such as electrochemical measurements [3] and energy-dispersive X-ray spectroscopy (EDX). [12]

The benefit of LP EM is the ability to study samples that do not withstand a vacuum or to study materials properties and reactions requiring liquid conditions. Examples of measurements enabled by this technique are the growth of metallic nanoparticles or structures in liquid, [13] [14] [15] [16] materials changes during the cycling of batteries, [8] [17] [18] electrochemical processes such as metal deposition, [3] dynamics of thin water films and diffusion processes, [19] biomineralization processes, [20] protein dynamics and structure, [21] [22] single-molecule localization of membrane proteins in mammalian cells, [4] [23] and the influence of drugs on receptors in cancer cells. [24]

The spatial resolution achievable can be in the sub-nanometer range and depends on the sample composition, structure and thickness, any window materials present, and the sensitivity of the sample to the electron dose required for imaging. [9] Nanometer resolution is obtained even in micrometers-thick water layers for STEM of nanomaterials of high atomic number. [4] [25] Brownian motion was found to be highly reduced with respect to a bulk liquid. [26] STEM detection is also possible in ESEM for imaging nanomaterials and biological cells in liquid. [27] [23] An important aspect of LP EM is the interaction of the electron beam with the sample [28] since the electron beam initiates a complex sequence of radiolytic reactions in water. [29] Nevertheless, quantitative analysis of LP EM data has yielded unique information in a range of scientific areas. [30] [31]

Related Research Articles

<span class="mw-page-title-main">Electron microscope</span> Type of microscope with electrons as a source of illumination

An electron microscope is a microscope that uses a beam of electrons as a source of illumination. They use electron optics that are analogous to the glass lenses of an optical light microscope. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light, electron microscopes have a higher resolution of about 0.1 nm, which compares to about 200 nm for light microscopes. Electron microscope may refer to:

<span class="mw-page-title-main">Scanning electron microscope</span> Type of electron 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 intensity of the detected signal to produce an image. In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using a secondary electron detector. The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography. Some SEMs can achieve resolutions better than 1 nanometer.

<span class="mw-page-title-main">Transmission electron microscopy</span> Technique in microscopy

Transmission electron microscopy (TEM) 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 scintillator attached to a charge-coupled device.

<span class="mw-page-title-main">Atomic force microscopy</span> Type of microscopy

Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.

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).

<span class="mw-page-title-main">Electron backscatter diffraction</span> Scanning electron microscopy technique

Electron backscatter diffraction (EBSD) is a scanning electron microscopy (SEM) technique used to study the crystallographic structure of materials. EBSD is carried out in a scanning electron microscope equipped with an EBSD detector comprising at least a phosphorescent screen, a compact lens and a low-light camera. In this configuration, the SEM incident beam hits the tilted sample. As backscattered electrons leave the sample, they interact with the crystal's periodic atomic lattice planes and diffract according to Bragg's law at various scattering angles before reaching the phosphor screen forming Kikuchi patterns (EBSPs). EBSD spatial resolution depends on many factors, including the nature of the material under study and the sample preparation. Thus, EBSPs can be indexed to provide information about the material's grain structure, grain orientation, and phase at the micro-scale. EBSD is applied for impurities and defect studies, plastic deformation, and statistical analysis for average misorientation, grain size, and crystallographic texture. EBSD can also be combined with energy-dispersive X-ray spectroscopy (EDS), cathodoluminescence (CL), and wavelength-dispersive X-ray spectroscopy (WDS) for advanced phase identification and materials discovery.

<span class="mw-page-title-main">Scanning transmission electron microscopy</span> Instrument that produces images by scanning electrons across a sample

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 so that the sample is illuminated at each point 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.

<span class="mw-page-title-main">Transmission electron cryomicroscopy</span>

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.

<span class="mw-page-title-main">Focused ion beam</span> Device

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.

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.

<span class="mw-page-title-main">Environmental scanning electron microscope</span> Scanning electron microscope with a gaseous environment in the specimen chamber

The environmental scanning electron microscope (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. Although there were earlier successes at viewing wet specimens in internal chambers in modified SEMs, the ESEM with its specialized electron detectors and its differential pumping systems, to allow for the transfer of the electron beam from the high vacuum in the gun area to the high pressure attainable in its specimen chamber, make it a complete and unique instrument designed for the purpose of imaging specimens in their natural state. The instrument was designed originally by Gerasimos Danilatos while working at the University of New South Wales.

<span class="mw-page-title-main">Ptychography</span>

Ptychography is a computational method of microscopic imaging. It generates images by processing many coherent interference patterns that have been scattered from an object of interest. Its defining characteristic is translational invariance, which means that the interference patterns are generated by one constant function moving laterally by a known amount with respect to another constant function. The interference patterns occur some distance away from these two components, so that the scattered waves spread out and "fold" into one another as shown in the figure.

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.

<span class="mw-page-title-main">Single particle analysis</span>

Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses. Individual images of stained or unstained particles are very noisy, and so hard to interpret. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with sub-nanometer resolution and near-atomic resolution first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well. Single particle analysis can also be performed by induced coupled plasma mass spectroscopy (ICP-MS).

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.

<span class="mw-page-title-main">Cryogenic electron microscopy</span> Form of transmission electron microscopy (TEM)

Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution. This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination without the need for crystallization.

A probe tip is an instrument used in scanning probe microscopes (SPMs) to scan the surface of a sample and make nano-scale images of surfaces and structures. The probe tip is mounted on the end of a cantilever and can be as sharp as a single atom. In microscopy, probe tip geometry and the composition of both the tip and the surface being probed directly affect resolution and imaging quality. Tip size and shape are extremely important in monitoring and detecting interactions between surfaces. SPMs can precisely measure electrostatic forces, magnetic forces, chemical bonding, Van der Waals forces, and capillary forces. SPMs can also reveal the morphology and topography of a surface.

Ultrafast scanning electron microscopy (UFSEM) combines two microscopic modalities, Pump-probe microscopy and Scanning electron microscope, to gather temporal and spatial resolution phenomena. The technique uses ultrashort laser pulses for pump excitation of the material and the sample response will be detected by an Everhart-Thornley detector. Acquiring data depends mainly on formation of images by raster scan mode after pumping with short laser pulse at different delay times. The characterization of the output image will be done through the temporal resolution aspect. Thus, the idea is to exploit the shorter DeBroglie wavelength in respect to the photons which has great impact to increase the resolution about 1 nm. That technique is an up-to-date approach to study the dynamic of charge on material surfaces.

4D scanning transmission electron microscopy is a subset of scanning transmission electron microscopy (STEM) which utilizes a pixelated electron detector to capture a convergent beam electron diffraction (CBED) pattern at each scan location. This technique captures a 2 dimensional reciprocal space image associated with each scan point as the beam rasters across a 2 dimensional region in real space, hence the name 4D STEM. Its development was enabled by evolution in STEM detectors and improvements computational power. The technique has applications in visual diffraction imaging, phase orientation and strain mapping, phase contrast analysis, among others.

References

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