Immunoelectron microscopy

Immunology

1. IMMUNOELECTRON MICROSCOPY BY : MEGHNA, RITIKA B., SIMAR JOT

2. ELECTRON MICROSCOPY Electron microscopy (EM) is a technique used for obtaining high resolution images of biological and non-biological specimens . It is used to investigate the detailed structure of tissues, cells, organelles and macromolecular complexes.   Electron microscopy is used in conjunction with a variety of ancillary techniques (e.g. thin sectioning, immuno-labeling ).

3. S everal cellular events may be missed if conventional ultrastructural studies are not complemented with details concerning the subcellular localization of a wide range of specific proteins. Thus, immunoelectron microscopy emerges as a technique that links the information gap between biochemistry, molecular biology , and ultrastructural studies, by placing macromolecular functions within a cellular context. IMMUNOELECTRON MICROSCOPY Immunoelectron microscopy is one of the best methods for detecting and localizing proteins in cells and tissues. This procedure can be used on practically every unicellular and multicellular organism, and often provides unexpected insights into the structure-function associations. It uses transmission electron microscope for visualisation. These days scanning electron microscope is also use. This technique uses antibodies to detect the intracellular location of structures of particular proteins.Ultra thin sections are labeled with antibodies against the required antigen and then labeled with gold particles. Gold particles of different diameters enable two or more proteins to be studied simultaneously.

4. Immunogold labelling -  colloidal gold  particles are most often attached to secondary   antibodies   which are in turn attached to primary antibodies designed to bind a specific  antigen or other cell component. Gold is used for its high electron density  which increases electron scatter to give high contrast 'dark spots '. Immunostaining (immunohistochemistry)- staining technique of tissue section so that the cells can be visualized under the microscope. The structure and location of antigen can be easily detected. Immunofixation – used for identification of antibodies for specific antigens.

5. IMMUNOGOLD LABELING U sed for the identification, localization, and distribution of proteins, antigens, and other macromolecules of interest, at an ultrastructural level . Powerful technique for identifying active sites and the presence of biomarkers in the cells. A primary antibody is designed to bind onto a specific antigen in the cells.  Gold conjugated secondary antibody designed to bind to primary antibody. Gold probe with its excellent electron scattering property is an important element for immunohistochemistry in the electron microscope

6. Principles of immunogold labelling USES OF THE IMMUNOGOLD TECHNIQUES 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. ANTIGEN-ANTIBODY REACTIONS Immunogold labeling is focused more on indirect patterns (gold conjugated secondary antibodies bind with specific primary antibodies in a microenvironment)   Indirect pattern is more favorable than the direct pattern for two reasons: higher density of secondary antibody and increased sensitivity, since the secondary antibody is able to bind with multiple sites on primary antibody

7. Success of immunogold labeling technique depends on – Quality of protein antigen preservation Antigen-primary antibody specificity Antibody’s ability to infiltrate cells and tissues.

8. GOLD PARTICLES AS A PROBE Gold became the most reliable choice for immunogold labeling due to L arge specific surface area G ood biocompatibility and H igh electron density, which offers easy detection The size of gold particles used for immunogold labeling varies from 1 to 40µm, chosen according to the type of labeling techniques employed Detecting multiple antigens within a cell may require the selective use of different sizes of gold particles . Smaller gold particles (2 nm or 5 nm) produce a higher labeling intensity and lower steric hindrance. Larger particle sizes (10 nm or more) reduce the potential labeling intensity due to their sheer size but are more easily seen at lower magnifications.

9. Pre-Embedding Immunogold Labeling is used primarily for the detection of proteins, antigens, and other macromolecules of interest, that are located on the surface or the exterior of cells, virus particles, and other extracellular biological specimens. In this technique specimens used are to be ultra-thin sectioned, for the examination of both the interior and exterior of cells, tissues, and other biological specimens. The different techniques normally used for immunogold labeling, dependent on the type of sample submitted and the location of the protein or macromolecule of interest. Post-Embedding Labeling techniques are used exclusively for the detection of proteins, antigens, and other macromolecules of interest that are located in the interior or intracellular regions of cells, virus particles, and other extracellular biological specimens.

10. APPLICATIONS OF IMMUNOELECTRON MICROSCOPY

11. Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes Electron Microscopy is an indispensable tool to investigate the intricate structures of the cell and organelles, and also to study the cellular biological processes implicated in the responses to changes in the microenvironment. However , several cellular events may be missed if conventional ultra structural studies are not complemented with details concerning the subcellular localization of a wide range of specific proteins which can become rearranged as part of their own dynamic processes. Thus , immunoelectron microscopy emerges as a technique that links the information gap between biochemistry, molecular biology, and ultra structural studies, by placing macromolecular functions within a cellular context. Furthermore , at the ultra structural level, we demonstrate the role of immunogold labeling in the study of biological processes induced by different stimuli from the environment.

12. Immunoelectron Microscopy of Parasites Immunoelectron Microscopy is a powerful tool for studying host–parasite interactions, and it is playing an important role in identifying specific immune targets and characterizing the precise subcellular localization, transport, and expression of parasite antigens. This technique helps to clarify specific functions of subcellular organelles, which may not otherwise be detected by standard electron microscopy or biochemical techniques. So , Immunoelectron Microscopy contributes to a better understanding of the relationship between structure and function in parasites. In studies of  Plasmodium ,   Immunoelectron Microscopy has been, especially valuable in characterizing the antigenic composition of intracellular compartments, for example, parasitophorous vacuole and cytoplasmic clefts, that cannot be isolated, purified, and studied by current biochemical procedures.

13. Immunoelectron microscopy techniques in plant virus diagnosis . The detection of virus in samples using electron microscopy can be enhanced by the use of specific antibodies to trap particles. Antibodies can further be used to label immobilized particles on grids to aid their identification.

14. Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen, immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.

15. Developments in cell biology for quantitative immunoelectron microscopy Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments . Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures . The methods can be used to analyze gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments. For volume-occupiers, Labelling Density (LD) can be expressed simply as gold's per test point and, for surface-occupiers, as gold's per test line intersection . Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions.

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