3. Light microscopy
• Light microscopy can reveal:
Characteristic inclusion bodies or
multinucleated giant cells
Location of inclusion bodies are diagnostic
• Inclusion bodies in nucleus------ herpes virus
• Inclusion bodies in cytoplasm------ pox virus
• Example
• Tzanck smear ------ herpes virus induced multinucleated giant cells in
vesicular skin lesions
• Owl’s eye shaped inclusion bodies------ cytomegalovirus infected cells
4.
5. Electron microscope
• Detects virus particles
• Morphology (capsid symmetry) and
• size aids in identification
Immunoelectron microscopy
• Antibody attached to viral antigens within tissue specimens
6. SEM: Avian flu virus
TEM reveals Rubella virions in the
process of budding from the host
cell surface
7. EM in Surveillance of Emerging Diseases
Viral emerging diseases identified first by EM
Parvovirus B-19
Monkeypox
SARS coronavirus
Metapneumovirus
Morbilivirus
Nipah virus
8. Advantages of EM in Virus Diagnosis/Identification
• Fast
• • Does not require living organisms
• Does not require special reagents (antibodies, nucleic acid standards,
protein standards)
• Works when other modalities are not enough (mutant genomes may
not react in PCRs)
• No false positive results (cross-reactions with similar things)
• What you see is the real thing
9. Limitations of EM in Virus Diagnosis/Identification
• Requires expensive instruments (EM)
• Requires virology knowledge
10. UV Microscopy
• UV microscopy is used when the specimens are treated with
fluorescent dyes
• Fluorescence microscopy tracks viral DNA in cells
13. Introduction
• “Immunofluorescence ( IF ) is a technique used for light microscopy
with a fluorescence microscope and is used primarily on
microbiological samples.”
• This method is focused on immune response that occurs in disease
tissues or cells
13
14. • Most widely it is used to visualize the fluorophore attached
antibodies binding with protein, glycoproteins and other antigenic
molecules.
• It is widely used example of Immunostaining and
immunohistochemistry
15. Principle
• Property of fluorophores to absorb light of certain wavelength and
change into “excited “state and emission of different wavelength on
coming back to “ground” state
15
17. Fluorophore
• These are the molecules that are used in fluorescent microscopy and
they have ability to absorb light of one wavelength and can send light
of different wavelength
• Most commonly used fluorophores are
• Isothyocyanate— Green
• Rhodamine— Orange
• Texas red— Red
17
18. Types of Immunofluorescence
1. Direct immunofluorescence(IF)
• fluorescent dye is attached to antibodies and use to detect antigen in sample
• Antigen is attached to slide
• Fluorescent attached antibodies are poured over it
• Washing is done to remove unattached Abs
• Examine under fluorescent microscope
18
19. 2. Indirect immunofluorescence(IF)
• Antibodies are applied to the attached antigen
• Fluorophore attached antibodies are targeted against antibodies
• Observed under fluorescent microscope
19
21. Advantages and disadvantages
• Direct IF reduces time and reduces number of steps and reduce non-
specific background signals
• They limit the possibility of cross reactivity
• Direct IF is less sensitive than Indirect IF and may result in False negative
• Indirect IF is more complex and time consuming but allow more flexibility
21
23. Limitations
• Photo bleaching— Fading of fluorophore due to breakage of covalent
bond or non specific reactions of fluorophore with surrounding
molecules
• Non specific binding can cause false positive and false negative
23
24. Applications Of Immunofluorescence
• It can be performed on biopsy materials of different tissue parts
Skin
Muscle
Lungs
• To detect auto-antibody from serum and other body fluids
• Microbiology
o Bacterial
o Viral
24
Fig. 1: This chart illustrates a typical workflow of indirect IF with epithelial cells adherently growing on coverslips. After cultivation, cells get fixed and therefore killed with a chemical crosslinker (e.g. formaldehyde). Next a permeabilization step with detergents is performed to enable the antibodies to cross the cellular membranes. Blocking with normal serum, milk powder or bovine serum albumin reduces the unspecific binding of antibodies to non-target structures in order to minimize false-positive signals. Next the incubation with the first antibody takes place, which specifically recognizes epitopes on the target molecule. In a second incubation step the fluorescence-coupled secondary antibody is applied which binds to the first antibody and therefore visualizes the target structure. After antibody incubation, nuclei staining is performed with dyes such as DAPI or Hoechst which intercalate into DNA. After mounting of the coverslip with a mounting medium (e.g. Mowiol® or Vectashield®) on a microscope slide, the IF preparation is ready for microscopy.
