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Structural biology is a field that is many centuries old which, as defined by the Journal of Structural Biology, deals with structural analysis of living material at every level of organization. Early structural biologists throughout the 19th and early 20th centuries were primarily only able to study structures to the limit of the naked eye's visual acuity and through magnifying glasses and light microscopes.
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.
Electron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope (TEM). It can involve the use of high-resolution transmission electron microscopy images, electron diffraction patterns including convergent-beam electron diffraction or combinations of these. It has been successful in determining some bulk structures, and also surface structures. Two related methods are low-energy electron diffraction which has solved the structure of many surfaces, and reflection high-energy electron diffraction which is used to monitor surfaces often during growth.
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.
A single-molecule experiment is an experiment that investigates the properties of individual molecules. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior of molecules cannot be distinguished, and only average characteristics can be measured. Since many measurement techniques in biology, chemistry, and physics are not sensitive enough to observe single molecules, single-molecule fluorescence techniques caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past. Indeed, since the 1990s, many techniques for probing individual molecules have been developed.
Electron cryotomography (cryo-ET) is an imaging technique used to produce high-resolution (~1–4 nm) three-dimensional views of samples, often biological macromolecules and cells. cryo-ET 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 imaged under cryogenic conditions. For cellular material, the structure is immobilized in non-crystalline, vitreous ice, allowing them to be imaged without dehydration or chemical fixation, which would otherwise disrupt or distort biological structures.
Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy and cryo-electron microscopy. Typical specimens for cryofixation include small samples of plant or animal tissue, cell suspensions of microorganisms or cultured cells, suspensions of viruses or virus capsids and samples of purified macromolecules, especially proteins.
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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.
Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction. A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because light sheet fluorescence microscopy scans samples by using a plane of light instead of a point, it can acquire images at speeds 100 to 1,000 times faster than those offered by point-scanning methods.
Robert Eric Betzig is an American physicist who works as a professor of physics and professor of molecular and cell biology at the University of California, Berkeley. He is also a senior fellow at the Janelia Farm Research Campus in Ashburn, Virginia.
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Scanning electron cryomicroscopy (CryoSEM) is a form of electron microscopy where a hydrated but cryogenically fixed sample is imaged on a scanning electron microscope's cold stage in a cryogenic chamber. The cooling is usually achieved with liquid nitrogen. CryoSEM of biological samples with a high moisture content can be done faster with fewer sample preparation steps than conventional SEM. In addition, the dehydration processes needed to prepare a biological sample for a conventional SEM chamber create numerous distortions in the tissue leading to structural artifacts during imaging.
Correlative light-electron microscopy (CLEM) is the combination of an optical microscope - usually a fluorescence microscope - with an electron microscope. In an integrated CLEM system, the sample is imaged using an electron beam and an optical light path simultaneously. Traditionally, samples would be imaged using two separate microscopy modalities, potentially at different facilities and using different sample preparation methods. Integrated CLEM is thus considered to be beneficial because the methodology is quicker and easier, and it reduces the chance of changes in the sample during the process of data collection. Overlay of the two images is thus performed automatically as a result of the integration of two microscopes.
Jacques Dubochet is a retired Swiss biophysicist. He is a former researcher at the European Molecular Biology Laboratory in Heidelberg, Germany, and an honorary professor of biophysics at the University of Lausanne in Switzerland.
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.
References
- ↑ Kumai, Motoi (1951-06-01). "Electron-Microscope Study of Snow-Crystal Nuclei". Journal of the Atmospheric Sciences. 8 (3): 151–156. doi: 10.1175/1520-0469(1951)008<0151:EMSOSC>2.0.CO;2 . ISSN 1520-0469.
- ↑ Cressey, Daniel; Callaway, Ewen (2017-10-01). "Cryo-electron microscopy wins chemistry Nobel". Nature. 550 (7675): 167. doi: 10.1038/nature.2017.22738 . ISSN 1476-4687. PMID 29022937. S2CID 205252059.
- ↑ Sazaki, Gen; Zepeda, Salvador; Nakatsubo, Shunichi; Yokoyama, Etsuro; Furukawa, Yoshinori (2010-11-16). "Elementary steps at the surface of ice crystals visualized by advanced optical microscopy". Proceedings of the National Academy of Sciences. 107 (46): 19702–19707. doi: 10.1073/pnas.1008866107 . ISSN 0027-8424. PMC 2993344 . PMID 20974928.
- ↑ Katakura, I.; Tokunaga, M.; Matsuo, A.; Kawaguchi, K.; Kindo, K.; Hitomi, M.; Akahoshi, D.; Kuwahara, H. (2010-04-12). "Development of high-speed polarizing imaging system for operation in high pulsed magnetic field". Review of Scientific Instruments. 81 (4): 043701. doi:10.1063/1.3359954. ISSN 0034-6748. PMID 20441339.
- ↑ Hulleman, Christiaan N.; Huisman, Maximiliaan; Moerland, Robert J.; Grünwald, David; Stallinga, Sjoerd; Rieger, Bernd (2018). "Fluorescence Polarization Control for On–Off Switching of Single Molecules at Cryogenic Temperatures". Small Methods. 2 (9): 1700323. doi:10.1002/smtd.201700323. ISSN 2366-9608. PMC 6592266 . PMID 31240238.
- ↑ Möckl, Leonhard; Lamb, Don C.; Bräuchle, Christoph (2014-12-15). "Super-resolved Fluorescence Microscopy: Nobel Prize in Chemistry 2014 for Eric Betzig, Stefan Hell, and William E. Moerner". Angewandte Chemie International Edition. 53 (51): 13972–13977. doi:10.1002/anie.201410265. PMID 25371081.