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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.
Spectroscopy is the study of the interaction between matter and electromagnetic radiation as a function of the wavelength or frequency of the radiation. In simpler terms, spectroscopy is the precise study of color as generalized from visible light to all bands of the electromagnetic spectrum; indeed, historically, spectroscopy originated as the study of the wavelength dependence of the absorption by gas phase matter of visible light dispersed by a prism. Matter waves and acoustic waves can also be considered forms of radiative energy, and recently gravitational waves have been associated with a spectral signature in the context of the Laser Interferometer Gravitational-Wave Observatory (LIGO).
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.
X-ray crystallography (XRC) is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their crystallographic disorder, and various other information.
Holography is a technique that enables a wavefront to be recorded and later re-constructed. Holography is best known as a method of generating three-dimensional images, but it also has a wide range of other applications. In principle, it is possible to make a hologram for any type of wave.
Raman spectroscopy ; is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.
X-ray fluorescence (XRF) is the emission of characteristic "secondary" X-rays from a material that has been excited by being bombarded with high-energy X-rays or gamma rays. The phenomenon is widely used for elemental analysis and chemical analysis, particularly in the investigation of metals, glass, ceramics and building materials, and for research in geochemistry, forensic science, archaeology and art objects such as paintings
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.
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.
An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations.
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.
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.
The Reverse Monte Carlo (RMC) modelling method is a variation of the standard Metropolis-Hastings algorithm to solve an inverse problem whereby a model is adjusted until its parameters have the greatest consistency with experimental data. Inverse problems are found in many branches of science and mathematics, but this approach is probably best known for its applications in condensed matter physics and solid state chemistry.
Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.
Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures of sub-cellular, macro-molecular, or materials specimens. 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.
Coherent diffractive imaging (CDI) is a "lensless" technique for 2D or 3D reconstruction of the image of nanoscale structures such as nanotubes, nanocrystals, porous nanocrystalline layers, defects, potentially proteins, and more. In CDI, a highly coherent beam of x-rays, electrons or other wavelike particle or photon is incident on an object.
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.
Phase-contrast X-ray imaging (PCI) or phase-sensitive X-ray imaging is a general term for different technical methods that use information concerning changes in the phase of an X-ray beam that passes through an object in order to create its images. Standard X-ray imaging techniques like radiography or computed tomography (CT) rely on a decrease of the X-ray beam's intensity (attenuation) when traversing the sample, which can be measured directly with the assistance of an X-ray detector. In PCI however, the beam's phase shift caused by the sample is not measured directly, but is transformed into variations in intensity, which then can be recorded by the detector.
Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.
Live-cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. Live-cell imaging was pioneered in first decade of the 21st century. One of the first time-lapse microcinematographic films of cells ever made was made by Julius Ries, showing the fertilization and development of the sea urchin egg. Since then, several microscopy methods have been developed to study living cells in greater detail with less effort. A newer type of imaging using quantum dots have been used, as they are shown to be more stable. The development of holotomographic microscopy has disregarded phototoxicity and other staining-derived disadvantages by implementing digital staining based on cells’ refractive index.
References
- ↑ Hayashi, Kouichi; Happo, Naohisa; Hosokawa, Shinya; Hu, Wen; Matsushita, Tomohiro (2012). "X-ray fluorescence holography". Journal of Physics: Condensed Matter. 24 (9): 093201. Bibcode:2012JPCM...24i3201H. doi:10.1088/0953-8984/24/9/093201. ISSN 0953-8984. PMID 22318258.
- ↑ Hayashi, K. (2003). "3D atomic imaging of SiGe system by X-ray fluorescence holography". Journal of Materials Science: Materials in Electronics. 14 (5/7): 459–462. doi:10.1023/A:1023993911437. ISSN 0957-4522. S2CID 134765278.
- ↑ Chukhovskii, F. N.; Poliakov, A. M. (2003). "X-ray fluorescence holography: a novel treatment for crystal structure determination". Acta Crystallographica Section A. 59 (2): 109–116. doi:10.1107/S0108767302022274. ISSN 0108-7673. PMID 12604848.
- ↑ Xie, Honglan; Chen, Jianwen; Gao, Hongyi; Xiong, Shisheng; Xu, Zhizhan (2004). "Removing twin images in X-ray fluorescence holography". Optics Communications. 229 (1–6): 123–129. Bibcode:2004OptCo.229..123X. doi:10.1016/j.optcom.2003.10.033. ISSN 0030-4018.
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