Immunoelectronmicroscopy principle and applications.pptx

Immunoelectron microscopy (IEM) allows the visualization of antigen-antibody interactions in an electron microscope (EM).
The technique was first described in 1941, using plant viruses and their antibodies, and has been employed in many biologic systems.
Immunoelectron microscopy can be defined as any technique that uses antibodies, or molecules that interact with antibodies (for example, protein A or protein G), in conjunction with electron microscopy to localize ultrastructurally antigens or antibodies in cells and tissues.
TRANSMISSION ELECTRON MICROSCOPE
In the transmission electron microscope, electrons serve as a pseudo-light source.
High-energy electrons emitted from a filament are accelerated and forced by electromagnets into a very fine coherent beam in the center of the microscope column.
Because electrons are used, the microscope column and specimen area are maintained in a vacuum (10−6–10−8 Torr) to prevent the deflection of electrons by atmospheric gases.
The electron beam is focused on to the sample.
Here, the more electron-dense molecules in the sample under observation adsorb and scatter electrons, preventing their passage through the sample.
Those electrons with enough energy to penetrate are transmitted, further focused, and projected on to a phosphor screen.
The image is resolved on the phosphor screen due to the emission of photons in the area struck by the transmitted electrons.
The image can be permanently recorded directly on photographic film sensitive to electrons or visualized with photographic cameras interfaced with the electron microscope viewing chamber and a computer to rapidly generate high-resolution computer processed images.
In preparation for transmission electron microscopy, samples are usually fixed with aldehydes (glutaraldehyde, formaldehyde or a combination of the two), post-aldehyde fixed in osmic acid, en bloc stained with a uranyl acetate solution, dehydrated with alcohols and embedded in a plastic resin.
The embedded samples are thin sectioned with a glass or diamond knife and subsequently counterstained with heavy metals (uranium and lead salts) to further enhance the electron density of positively or negatively charged molecules in the sample.
Specimens thus prepared have good contrast and can be used to observe and record the molecular and subcellular detail of cells and tissues.
Alternatively, molecules may be adsorbed to a thin plastic film followed by embedding in an electron-dense stain or angular shadowing with evaporated metals.
The mechanical principles of operation for the scanning electron microscope are virtually the same as for transmission electron microscopes.
The major difference is in the formation of the image.
In scanning electron microscopes, the focused electrons are scanned over the surface of a sample, as implied by the name.
Instead of transmitting electrons, secondary electrons emitted from the interaction of electron beam and sample are collected, electronically

1. IMMUNOELECTRON MICROSCOPY Rinchen Chalese MSc Medical Microbiology

2. Introduction Immunoelectron microscopy (IEM) allows the visualization of antigen-antibody interactions in an electron microscope (EM). The technique was first described in 1941, using plant viruses and their antibodies, and has been employed in many biologic systems. Immunoelectron microscopy can be defined as any technique that uses antibodies, or molecules that interact with antibodies (for example, protein A or protein G), in conjunction with  electron microscopy  to localize ultrastructurally antigens or antibodies in cells and tissues.

3. TRANSMISSION ELECTRON MICROSCOPE In the transmission electron microscope, electrons serve as a pseudo-light source. High-energy electrons emitted from a filament are accelerated and forced by electromagnets into a very fine coherent beam in the center of the microscope column. Because electrons are used, the microscope column and specimen area are maintained in a vacuum (10 −6 –10 −8  Torr) to prevent the deflection of electrons by atmospheric gases. The  electron beam  is focused on to the sample. Here, the more electron-dense molecules in the sample under observation adsorb and scatter electrons, preventing their passage through the sample. Those electrons with enough energy to penetrate are transmitted, further focused, and projected on to a phosphor screen. The image is resolved on the phosphor screen due to the emission of photons in the area struck by the transmitted electrons. The image can be permanently recorded directly on photographic film sensitive to electrons or visualized with photographic cameras interfaced with the electron microscope viewing chamber and a computer to rapidly generate high-resolution computer processed images.

4. In preparation for  transmission electron microscopy , samples are usually fixed with aldehydes (glutaraldehyde, formaldehyde or a combination of the two), post-aldehyde fixed in  osmic acid ,  en bloc  stained with a  uranyl acetate  solution, dehydrated with alcohols and embedded in a plastic resin. The embedded samples are thin sectioned with a glass or diamond knife and subsequently counterstained with  heavy metals  (uranium and lead salts) to further enhance the electron density of positively or negatively charged molecules in the sample. Specimens thus prepared have good contrast and can be used to observe and record the molecular and subcellular detail of cells and tissues. Alternatively, molecules may be adsorbed to a thin plastic film followed by embedding in an electron-dense stain or angular shadowing with evaporated metals.

5. SCANNING ELECTRON MICROSCOPY The mechanical principles of operation for the scanning electron microscope are virtually the same as for transmission electron microscopes. The major difference is in the formation of the image. In scanning electron microscopes, the focused electrons are scanned over the  surface  of a sample, as implied by the name. Instead of transmitting electrons,  secondary electrons  emitted from the interaction of electron beam and sample are collected, electronically deciphered and multiplied, and projected on to a high-resolution cathode-ray tube. These images give a quasi three-dimensional impression of the structure under observation because of the great depth of field of the scanning electron microscope. The image is recorded on film using an ordinary camera focused on the cathode-ray tube. Samples for scanning electron microscopy must be fixed and dehydrated, as in transmission electron microscopy, but instead of embedding the sample in a plastic resin, it is covered with a conductive coating of a fine metal, such as gold, by evaporation; no further processing is necessary. Resolution of TEM: 50pm, SEM: 1nm

6. History of Immunoelectron microscopy

7. Applications Immunogold labeling is being very useful in the localization of target markers in cells and tissues. Provides excellent insight with regards to structure–function relationships in the microenvironment of cells and tissues. Used in the study of protein distribution in cellular and extracellular components

8. The most commonly used antibodies belong to the immunoglobulin G class. Direct imaging of bound antibodies can be achieved with purified small isolated samples such as macromolecules or viruses, usually after negative staining or metal coating, in transmission or scanning electron microscopes. Figure 1 Direct imaging of monovalent antibody fragments (Fab) bound to a T-even bacteriophage by transmission electron microscopy. Fab molecules (arrowheads) against phage Tu-6 allow the detection of homologous epitopes at distinct sites on the tail fibres of the related phage Tu-46. The phage preparation was negatively stained with uranyl acetate. Bar, 0.1 m m.

9. For bulk specimens or thick samples which have to be sectioned or cleaved before examination in the electron microscope, only an indirect visualization of bound antibodies is possible. Therefore, antibodies have to be conjugated with electron-dense markers. Commonly used markers are particles such as colloidal gold of different sizes (1–40 nm), nanogold and ferritin, or enzymes such as horseradish peroxidase, which are located by electron dense reaction products (e.g. using diaminobenzidine as substrate). Particulate markers have the advantage that they can be counted directly and therefore allow for quantification of bound antibodies. Figure 2 Indirect imaging of the viral glycoprotein gp70 on the surface of a Friend murine leukaemia virus. The picture shows an ultrathin section of a ferritin-labelled virus particle in the extracellular space. The sample was fixed chemically and labelled in two steps, first with an antibody against gp70, followed by a secondary antibody against the primary antibody. The secondary antibody itself is conjugated to the electron-dense marker molecule ferritin. In the centre of the image, the central viral core can be seen inside the envelope. The dark dots surrounding the virus are the iron containing cores of the ferritin particles.

10. The detection of antigens of thick (bulk) samples is approached in two ways. The first approach involves the labelling of structures accessible on surfaces (e.g. on viruses, cells or cleaved samples) or of intracellular structures after permeabilization with detergents, followed by direct inspection in the scanning electron microscope or indirect visualization in the transmission electron microscope after sectioning or preparing a replica. This approach is also referred to as pre-embedding labelling technique. A different approach involves the labelling of samples that have already been thinly sectioned, also referred to as an on-section or post-embedding technique

11. Labelling Nonspecific binding of antibodies to the specimen has to be blocked by incubation with proteins (e.g. albumin, gelatin or milk powder) and/or detergents (Tween 20) before and during the immunolabelling procedure. For most purposes, the indirect method (two-step reaction) of immunodetection is applied: the bound specific antibody is detected by complexes of markers conjugated to secondary antibodies or protein A molecules. The advantage here is that these complexes can be used universally to detect any antibodies from the same species. The direct method (one-step reaction), where the marker is directly conjugated to the antibody, is used less frequently; for each reaction a specific conjugate has to be prepared, with the additional risk of loss of protein activity when adsorbed on to the gold surface. However, the resolution of immunolocalization is slightly better than with the indirect method.

12. Typical conditions for the antigen–antibody interaction during immunolabelling are: antibody concentration in the range of micrograms per millilitre (if the concentration of the specific antibody is unknown, a dilution series has to be made), an incubation time in the range of 5–30 min (if antigens on surfaces have to be labelled) or longer (if diffusion barriers exist, as in permeabilized specimens) at a temperature between 4 ℃ and 40 ℃ , and pH and ion concentrations similar to physiological conditions or even higher. A pH up to 8 and a sodium chloride concentration of up to 1 molL 2 1 may improve affinity and minimize nonspecific binding.

13. References Immuno-electron Microscopy Heinz Schwarz, Max Planck Institute for Developmental Biology, Tu¨bingen , Germany Heinz Hohenberg, Heinrich Pette Institute for Experimental Virology and Immunology, Hamburg, Germany Electron Microscopy, Immunological Applications Matthew A. Gonda