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.
4. Several 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.
5. 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.
6. IMMUNOGOLD LABELING
Used 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
7. 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:
(a) higher density of secondary antibody and
(b) increased sensitivity, since the secondary antibody is able to bind with multiple sites on primary antibody
8. Success of immunogold labeling technique depends on –
• Quality of protein antigen preservation
• Antigen-primary antibody specificity
• Antibody’s ability to infiltrate cells and tissues.
9.
10. GOLD PARTICLES AS A PROBE
Gold became the most reliable choice for immunogold labeling due to
Large specific surface area
Good biocompatibility and
High 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.
11. 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.
14. 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.
15. 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.
16. 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.
17. 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.
18. 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.
