Author- M. Jyothsna Swaroop Batch- 2021 Branch-Biotechnology

1. IMMUNOELECTRON
MICROSCOPY
By :
M. Jyothsna Swaroop
M.Tech 1st year

2.  Immunoelectron microscopy is a method used in electron microscopy for diagnosis of viral
infections
 This technique was first described in the 1940s using tobacco mosaic virus. Later in
1960s, June Almeida used it to identify viruses including rubella virus and in1970s Albert
Kapikian discovered noroviruses using this method
 Powerful technique used for identifying viral antigens and determining their structural
localization and organization within vaccines and Tissues
 This procedure can be used in every unicellular and multicellular organism to find the
structure-function associations
 Immunoelectron microscopy is founded upon the principles of transmission electron
microscopy (TEM). TEM is an imaging technique that relies on a high voltage electron beam
being passed through a specimen, resulting in an image created from the interaction between
the electrons and the sample.
INTRODUCTION

3. ELECTRON MICROSCOPY
How does it work?
I. Uses a beam of accelerated electrons as a source of
illumination.
II. Gives images of high resolution which can be
magnified in nanometres
III. The images are formed by controlled use of
electrons in a vacuum captured on a
phosphorescent screen
IV. Electron microscopy is used in conjunction with a
variety of additional trchniques (like thin sectioning
, immunolabelling)
V. There are two types of Electron Microscopes
I. Transmission Electron Microscopy (TEM)
II. Scanning Electron Microscopy (SEM)

5. IMMUNOELECTRON MICROSCOPY
• Immunoelectron microscopy is founded upon the principles of transmission electron microscopy
(TEM).
• TEM is an imaging technique that depends upon a high voltage electron beam being passed
through a specimen, resulting in an image created from the interaction between the electrons and
the sample
• Negative staining is a quick and accessible way to identify structural information about a sample of
particles in suspension
• Utilizes negative staining techniques for antigen detection and structural organization of samples
• Samples prepared for immunoelectron microscopy are ultrathin sections of tissue treated for
microscopy applications, but valuable information can also be obtained from immunogold labeling
of liquid samples of viral suspensions.
• Not only can these techniques be applied to studying of live viruses, but they are also useful for
characterizing viral components in vaccines, viral like particles (VLPs), proteins purified from
viruses, and other specimen relevant in modern virology applications

6.  The ultrathin specimen needed in TEM requires special protocols producing very hard blocks
that allow cutting of 50-70 nm thick sections.
 This can be attained by embedding the sample in special plastic resins that harden after
polymerization, or by rapidly freezing down the specimen in liquid nitrogen and cutting it at
ultracold temperature (cryo-ultramicrotomy).
 Three general methods are used, depending on the amount and the intracellular location of
the antigen and on the fixation technique:
i. cryo-ultramicrotomy
ii. pre-embedding
iii. post-embedding
 All three methods include fixation, labelling , blocking , sectioning and contrasting in different
order before TEM examination
Sample preparation:

7. Flow Chart for Immunoelectron Microscopy Methods

8. Detection
• Immunoelectron microscopy combines the ability of an antibody to specifically bind a protein
with the high spatial resolution of an electron microscope in order to determine the protein’s
location at subcellular level.
• The visualization is performed by TEM, which gives a simple two-dimensional projection of
the ultrathin sample down the optic axis.
• Detection of the antibody’s localization in the sample is possible by conjugating it with
colloidal gold particles of known diameter, which are thus distinctly visible on the transmitted
image of the sample.
• By using the tomography system available on modern electron microscopes, one can go
beyond the two dimensional projection and get accurate 3D information on the location of the
protein and with even higher resolution power

9. Electron micrograph of gold nanoparticles attached to rotaviruses. The
small dark circular objects are gold nanoparticles coated with
a monoclonal antibody specific for rotavirus protein VP6

10. Immuno-gold labelling
Immunogold labeling is a very powerful technique for identifying active sites and the presence of
biomarkers in the cells
It is probably the only technique that can probe the cells ultrastructurally because of the
processes devised to attach gold probes to secondary antibodies and consecutively to primary
antibody to reveal the presence of an antigen
Uses:
Immunogold labeling is being very useful in the localization of target markers in cells and
tissues. The high resolution of the technique could provide excellent insight with regards to
structure–function relationships in the microenvironment of cells and tissues. It can also be used
in the study of protein distribution in cellular and extracellular components
However, success of immunogold labeling techniques is very much dependent on the quality of
protein antigen preservation, antigen-primary antibody specificity, and the antibody’s ability to
infiltrate cells and tissues
Good handling of samples which involves fixation and dehydration as well as an appropriate
selection of embedding media are crucial for immunogold labelling analysis

11. Antigen-Antibody reaction:
The antibody binds with the antigen through a specific site called epitope. Immunogold labeling
relies solely on this antibody-antigen reaction in order to get intended results.
There are two types of antibodies: monoclonal and polyclonal antibody.
Monoclonal antibodies are derived from cellular reaction of plasma cells while polyclonal
antibodies are derived from the repetition of antigen stimulation.
Generally, immunogold labeling is focused more on indirect patterns whereby gold conjugated
secondary antibodies bind with specific primary antibodies in a microenvironment. This indirect
pattern is more favorable than the direct pattern for two reasons: (a) higher density of secondary
antibody albeit for longer incubation time and (b) increased sensitivity, since the secondary
antibody is able to bind with multiple sites on primary antibody
Gold particles as Probe:
Gold became the most reliable choice for immunogold labeling due to its large specific surface
area, good biocompatibility and high electron density, which offers easy detection, excellent
electrical and thermal conductivity in the electron microscope.
The size of gold particles used for immunogold labeling varies from 1 to 40µm and can be 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.

13. Applications of Immunoelectron microscopy
 Analysis of cellular processes: It is used to investigate the intrinsic structures of the
cell and cell organelles, and also used to examine the cellular biological processes
connected in the responses to the environmental changes
 To study Host-Parasite interations: clarifies the specific functions of subcellular
organelles , which might not be detected by standard electron microscope or
biochemical techniques
 In plant-virus diagnosis
 In studies of nervous –system : focuses primarily on identifying the cellular and
subcellular localization of proteins of relevance to neurotransmission and on defining
synaptic connectivity within neuronal circuits