Structural Biochemistry/Bioimaging

Light microscopy

In light microscopy, glass lenses are used to focus a beam of light on to the specimen under investigation. The light passing through the specimen is then focused by other lenses to produce a magnified image. Standard (bright-field) light microscopy is the most common microscopy technique in use today and uses a compound microscope. The specimen is illuminated from underneath by a lamp in the base of the microscope, with the light being focused on to the plane of the specimen by a condenser lens. Incident light coming through the specimen is picked up by the objective lens and focused on to its focal plane, creating a magnified image. This image is further magnified by the eyepiece, with the total magnification achieved being the sum of the magnifications of the individual lenses. In order to increase the resolution achieved by a compound microscope, the specimen is often overlaid with immersion oil into which the objective lens is placed. The limit of resolution of the light microscope using visible light is approximately 0.2*10^-12m.

Fluorescence microscopy

In fluorescence microscopy, the light microscope is adapted to detect the light emitted by a fluorescent compound that is used to stain selectively components within the cell. A chemical is said to be fluorescent if it absorbs light at one wavelength (the excitation wavelength) and then emits light at a longer wave length (the emission wavelength). Two commonly used compounds in fluorescent microscopy are rhodamine and Texas red, which emit red light, and fluorescein, which emits green light. First, an antibody against the antigen of interest (so-called primary antibody) is added to the specimen. A fluorescent compound is chemically coupled to a secondary antibody that recognized the primary antibody. Then the fluorescently-tagged secondary antibody is added to the tissue section or permeabilized cell, and the specimen is illuminated with light at the exciting wavelength. The structures in the specimen to which the antibody has bound can then be visualized. Fluorescence microscopy can also be applied to living cells, which allows the movement of the cells and structures within them to be followed with time.

Green fluorescent protein

The discovery of a naturally fluorescent protein found in the jellyfish Aquorea victoria. In this 238 amino acid protein, called green fluorescent protein (GFP), certain amino acid side-chains has spontaneously cyclized to form a green-fluorescing chromophore. Using recombinant DNA techniques, the DNA encoding GFP can be tagged on to the DNA sequences encoding other proteins, and then introduced into living cells in culture or into specific cells of a whole animal. Cells containing the introduced gene will then produce the protein tagged with GFP which will fluoresce green under the fluorescent microscope. The localization and movement of the GFP-tagged protein can then be studied in living cells in real time. Multiple variations of GFP have been engineered which emit light at different wavelength. They allows several proteins to be visualized simultaneously in the same cell.

Transmission electron microscopy

In contrast with light microscopy where optical lenses focus a beam of light, in electron microscopy electromagnetic lenses focus a beam of electrons. Because electrons are absorbed by atoms in the air, the specimen has to be mounted in a vacuum within an evacuated tube. The resolution of the electron microscope with biological materials is at best 0.10 nm. In transmission electron microscopy, a beam of electron is directed through the specimen and electron magnetic lenses are used to focus the transmitted electrons to produce an image either on a viewing screen or on photographic film. As in standard light microscopy, thin sections of the specimen are viewed. However, for transmission electron microscopy the sections must be much thinner (50-100 nm thick). Since electrons pass uniformly through biological material, unstained specimens give very poor images.Therefore, the specimen must routinely be stained in order to scatter some of the incident electrons which are then not focused by the electromagnetic lenses and so do not form the image. Heavy metals such as gold and osmium are often used to stain biological materials. In particular osmium tetroxide preferentially stains certain cellular components, such as membranes, which appear black in the image. The transmission electron microscope has sufficiently high resolution that it can be used to obtain information about the shapes of purified proteins,viruses and subcellular organelles. Antibodies can be tagged with electron-dense gold particles in a similar way to being tagged with a fluorescent compound in fluorescence microscopy, and then bound to specific target proteins in the thin sections of the specimen. When viewed in the electron microscope, small dark spots due to the gold particles are seen in the image wherever an antibody molecules has bound to its antigen and so the technique can be used to localize specific antigens.

Scanning electron microscopy

In scanning electron microscopy, an (unsectioned) specimen is fixed and then coated within a thin layer of a heavy metal such as platinum.An electron beam then scans over the specimen, exciting molecules within it that release secondary electrons. These secondary electrons are focused on to a scintillation detector and the resulting image displayed on a cathode-ray tube. The scanning electron microscope produces a three-dimensional image because the number of secondary electrons produced by any one point on the specimen depends on the angel of the electron beam in relation to the surface of the specimen. The resolution of the scanning electron microscope is 10 nm, some 100-fold less than that of the transmission electron microscope.

Reference

David Hanes,Nigel Hooper.Biochemistry. Taylor and Francis Group. New York. 2005.