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.
Ionization or ionisation is the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons, often in conjunction with other chemical changes. The resulting electrically charged atom or molecule is called an ion. Ionization can result from the loss of an electron after collisions with subatomic particles, collisions with other atoms, molecules and ions, or through the interaction with electromagnetic radiation. Heterolytic bond cleavage and heterolytic substitution reactions can result in the formation of ion pairs. Ionization can occur through radioactive decay by the internal conversion process, in which an excited nucleus transfers its energy to one of the inner-shell electrons causing it to be ejected.
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.
Optical coherence tomography (OCT) is an imaging technique that uses low-coherence light to capture micrometer-resolution, two- and three-dimensional images from within optical scattering media. It is used for medical imaging and industrial nondestructive testing (NDT). Optical coherence tomography is based on low-coherence interferometry, typically employing near-infrared light. The use of relatively long wavelength light allows it to penetrate into the scattering medium. Confocal microscopy, another optical technique, typically penetrates less deeply into the sample but with higher resolution.
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).
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), 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.
Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique based on the differences in the exponential decay rate of the fluorophore from a sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.
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.
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy.
Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was, perhaps, unknown to Hell and Wichmann in 1994.
Second-harmonic imaging microscopy (SHIM) is based on a nonlinear optical effect known as second-harmonic generation (SHG). SHIM has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. A second-harmonic microscope obtains contrasts from variations in a specimen's ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in optical density, path length, or refractive index of the specimen. SHG requires intense laser light passing through a material with a noncentrosymmetric molecular structure. Second-harmonic light emerging from an SHG material is exactly half the wavelength (frequency doubled) of the light entering the material. While two-photon-excited fluorescence (TPEF) is also a two photon process, TPEF loses some energy during the relaxation of the excited state, while SHG is energy conserving. Typically, an inorganic crystal is used to produce SHG light such as lithium niobate (LiNbO3), potassium titanyl phosphate (KTP = KTiOPO4), and lithium triborate (LBO = LiB3O5). Though SHG requires a material to have specific molecular orientation in order for the incident light to be frequency doubled, some biological materials can be highly polarizable, and assemble into fairly ordered, large noncentrosymmetric structures. Biological materials such as collagen, microtubules, and muscle myosin can produce SHG signals. The SHG pattern is mainly determined by the phase matching condition. A common setup for an SHG imaging system will have a laser scanning microscope with a titanium sapphire mode-locked laser as the excitation source. The SHG signal is propagated in the forward direction. However, some experiments have shown that objects on the order of about a tenth of the wavelength of the SHG produced signal will produce nearly equal forward and backward signals.
Christoph Cremer is a German physicist and emeritus at the Ruprecht-Karls-University Heidelberg, honorary professor at the University of Mainz and was a former group leader at the Institute of Molecular Biology (IMB) at the Johannes Gutenberg University of Mainz, Germany, who has successfully overcome the conventional limit of resolution that applies to light based investigations by a range of different methods.
Multiphoton lithography of polymer templates has been known for years by the photonic crystal community. Similar to standard photolithography techniques, structuring is accomplished by illuminating negative-tone or positive-tone photoresists via light of a well-defined wavelength. The fundamental difference is, however, the avoidance of reticles. Instead, two-photon absorption is utilized to induce a dramatic change in the solubility of the resist for appropriate developers.
Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
A multifocal diffractive lens is a diffractive optical element (DOE) that allows a single incident beam to be focused simultaneously at several positions along the propagation axis.
The Beckman Laser Institute is an interdisciplinary research center for the development of optical technologies and their use in biology and medicine. Located on the campus of the University of California, Irvine in Irvine, California, an independent nonprofit corporation was created in 1982, under the leadership of Michael W. Berns, and the actual facility opened on June 4, 1986. It is one of a number of institutions focused on translational research, connecting research and medical applications. Researchers at the institute have developed laser techniques for the manipulation of structures within a living cell, and applied them medically in treatment of skin conditions, stroke, and cancer, among others.
Wide-field multiphoton microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 µm). The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy. Wide-field multiphoton microscopes are not yet commercially available, but working prototypes exist in several optics laboratories.
Photoacoustic microscopy is an imaging method based on the photoacoustic effect and is a subset of photoacoustic tomography. Photoacoustic microscopy takes advantage of the local temperature rise that occurs as a result of light absorption in tissue. Using a nanosecond pulsed laser beam, tissues undergo thermoelastic expansion, resulting in the release of a wide-band acoustic wave that can be detected using a high-frequency ultrasound transducer. Since ultrasonic scattering in tissue is weaker than optical scattering, photoacoustic microscopy is capable of achieving high-resolution images at greater depths than conventional microscopy methods. Furthermore, photoacoustic microscopy is especially useful in the field of biomedical imaging due to its scalability. By adjusting the optical and acoustic foci, lateral resolution may be optimized for the desired imaging depth.
Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength laser to excite the fluorescent dyes with three simultaneously absorbed photons, and then the fluorescent dyes emit one photon whose energy is three times the energy of each incident photon. Comparing to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plan by , which is much faster than that of two-photon microscopy. In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect, which causes three photon microscopy to have higher resolution than conventional microscopy.
Ultrafast scanning electron microscopy (UFSEM) is an innovative consolidated facility that combines two microscopic modalities, Pump-probe microscopy and Scanning electron microscope, to gather temporal and spatial resolution phenomena. In fact, this technique is very wonderful at which ultrashort laser will be used 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.
