In microscopy, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with positive staining, in which the actual specimen is stained.
For bright-field microscopy, negative staining is typically performed using a black ink fluid such as nigrosin and India ink. The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark surrounding background. An alternative method has been developed using an ordinary waterproof marking pen to deliver the negative stain. [1]
In the case of transmission electron microscopy, opaqueness to electrons is related to the atomic number, i.e., the number of protons. Some suitable negative stains include ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osmium ferricyanide [2] and auroglucothionate. These have been chosen because they scatter electrons strongly and also adsorb to biological matter well. The structures which can be negatively stained are much smaller than those studied with the light microscope. Here, the method is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power. Some stains, such as osmium tetroxide and osmium ferricyanide, are very chemically active. As strong oxidants, they cross-link lipids mainly by reacting with unsaturated carbon-carbon bonds, and thereby both fix biological membranes in place in tissue samples and simultaneously stain them. [3] [4]
The choice of negative stain in electron microscopy can be very important. An early study of plant viruses using negatively stained leaf dips from a diseased plant showed only spherical viruses with one stain and only rod-shaped viruses with another. The verified conclusion was that this plant suffered from a mixed infection by two separate viruses. Negative staining at both light microscope and electron microscope level should never be performed with infectious organisms unless stringent safety precautions are followed. Negative staining is usually a very mild preparation method and thus does not reduce the possibility of operator infection.
Negative staining transmission electron microscopy has also been successfully employed for study and identification of aqueous lipid aggregates like lamellar liposomes (le), inverted spherical micelles (M) and inverted hexagonal HII cylindrical (H) phases (see figure). [5]
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 10,000,000× whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000×.
Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.
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 a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being 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 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.
Osmium tetroxide (also osmium(VIII) oxide) is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite its toxicity and the rarity of osmium. It also has a number of unusual properties, one being that the solid is volatile. The compound is colourless, but most samples appear yellow. This is most likely due to the presence of the impurity OsO2, which is yellow-brown in colour.
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.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of disease at a microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in gram-negative bacteria. Using cryo-electron microscopy it has been found that a much smaller periplasmic space is also present in gram-positive bacteria.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
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.
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.
Dark-field microscopy describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen is generally dark.
Polymorphism in biophysics is the ability of lipids to aggregate in a variety of ways, giving rise to structures of different shapes, known as "phases". This can be in the form of sphere of lipid molecules (micelles), pairs of layers that face one another, a tubular arrangement (hexagonal), or various cubic phases. More complicated aggregations have also been observed, such as rhombohedral, tetragonal and orthorhombic phases.
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.
The transmission electron microscope (TEM) is used as an important diagnostic tool to screen human tissues at high magnification, often in conjunction with other methods, particularly light microscopy and immunofluorescence techniques. The TEM was first used extensively for this purpose in the 1980s, especially for identifying the markers of cell differentiation to identify tumours, and in renal disease. Immunolabelling techniques are now generally used instead of the TEM for tumour diagnosis but the technique retains a critical role in the diagnosis of renal disease and a range of other conditions.
Lipid bilayer characterization is the use of various optical, chemical and physical probing methods to study the properties of lipid bilayers. Many of these techniques are elaborate and require expensive equipment because the fundamental nature of the lipid bilayer makes it a very difficult structure to study. An individual bilayer, since it is only a few nanometers thick, is invisible in traditional light microscopy. The bilayer is also a relatively fragile structure since it is held together entirely by non-covalent bonds and is irreversibly destroyed if removed from water. In spite of these limitations dozens of techniques have been developed over the last seventy years to allow investigations of the structure and function of bilayers. The first general approach was to utilize non-destructive in situ measurements such as x-ray diffraction and electrical resistance which measured bilayer properties but did not actually image the bilayer. Later, protocols were developed to modify the bilayer and allow its direct visualization at first in the electron microscope and, more recently, with fluorescence microscopy. Over the past two decades, a new generation of characterization tools including AFM has allowed the direct probing and imaging of membranes in situ with little to no chemical or physical modification. More recently, dual polarisation interferometry has been used to measure the optical birefringence of lipid bilayers to characterise order and disruption associated with interactions or environmental effects.
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.
Transmission electron microscopy DNA sequencing is a single-molecule sequencing technology that uses transmission electron microscopy techniques. The method was conceived and developed in the 1960s and 70s, but lost favor when the extent of damage to the sample was recognized.
Lamellar phase refers generally to packing of polar-headed long chain nonpolar-tail molecules in an environment of bulk polar liquid, as sheets of bilayers separated by bulk liquid. In biophysics, polar lipids pack as a liquid crystalline bilayer, with hydrophobic fatty acyl long chains directed inwardly and polar headgroups of lipids aligned on the outside in contact with water, as a 2-dimensional flat sheet surface. Under transmission electron microscope (TEM), after staining with polar headgroup reactive chemical osmium tetroxide, lamellar lipid phase appears as two thin parallel dark staining lines/sheets, constituted by aligned polar headgroups of lipids. 'Sandwiched' between these two parallel lines, there exists one thicker line/sheet of non-staining closely packed layer of long lipid fatty acyl chains. This TEM-appearance became famous as Robertson's unit membrane - the basis of all biological membranes, and structure of lipid bilayer in unilamellar liposomes. In multilamellar liposomes, many such lipid bilayer sheets are layered concentrically with water layers in between.
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