Want to learn about immunofluoresence? This presentation will go over some basic and popular immunofluoresence concepts in a concise fashion. Featuring:
Introduction
History
Similarities & Difference between IF and IHC
Types of Immunofluorescence
Popular Terms
Commonly used Fluorophores
Disease Diagnosed by Immunofluorescence
Antibodies, Proteins and Genes associated with Immunofluorescence
2. Outline
• Introduction
• History
• Similarities & Difference between IF and IHC
• Types of Immunofluorescence
• Popular Terms
• Commonly used Fluorophores
• Disease Diagnosed by Immunofluorescence
• Antibodies, Proteins and Genes associated with
Immunofluorescence
3. Introduction
What is Immunofluorescence?
It is a histochemical laboratory staining technique that
relies on antibodies–antigens interactions. It uses
antibodies' antigen specificity to direct fluorescent dyes to
particular molecule targets within a cell, allowing
visualization of the target molecule's distribution within the
sample.
4. Brief History of Immunofluorescence
• In 1941, Albert Hewett Coons and colleagues were able to
visualize pneumococcal antigens in infected tissue
specimens using fluorescently labeled antibodies. The
findings were celebrated as a watershed moment, and the
procedure has been endlessly developed, modified, and
refined since then, resulting in an undeniably useful and
flexible tool for both diagnostic and research laboratories.
• Immunofluorescence can be described also as a type of
IHC, wherein IF Monoclonal and polyclonal antibodies are
analyzed using a fluorescence detection system, while IHC
uses chemical-based approaches to detect monoclonal and
polyclonal antibodies.
5. Similarities Between IF And IHC
1. Both takes place under in-vitro
conditions
2. Require antigen-antibody interaction
3. They have reproducible results
4. They have improved data quality
5. The techniques are rapid
6. Both can be used in diagnosis of
cancers and infectious diseases
6. Differences between IF and IHC
Immunofluorescence (IF) Immunohistochemistry (IHC )
IF is a method of identification in which
fluorescent dyes or fluorescent proteins are used
to mark the antibodies used in the assay.
IHC is a method of identification in which the
antibodies in an assay are labelled with chemicals
or radioactive elements.
IF always has a high accuracy than IHC IHC has lower accuracy relative to IF
There is high specificity in IF There is less specificity compared to IF
7. Why Choose IF Over IHC?
• IHC is widely used to differentiate between
distinct cell groups that do not overlap. IF is
the staining method of choice for
multiplexing, especially when co-localization is
desired or when more than 2 different
antibodies are needed. With IF staining,
antibodies against specific proteins are added
to the slide and are then visualized with
fluorescent dyes and viewed with a
fluorescent microscope.
8. Types of Immunofluorescence
Direct Immunofluorescence
Direct IF involves use of a
single antibody that is aimed
towards the desired target
.The fluorophore is
specifically conjugated to
the main antibody. The
fluorophore it carries can be
detected via microscopy.
Indirect Immunofluorescence
Indirect IF uses two
antibodies. The primary
antibody is unconjugated and
a fluorophore-conjugated
secondary antibody directed
against the primary antibody is
used for detection.
9. Differences Between Direct And
Indirect Immunofluorescence
Direct Immunofluorescence Indirect Immunofluorescence
Relative shorter protocol, due to absence of
Secondary antibody incubation.
Takes a longer time, due to increased steps for
Secondary antibodies.
Less background due to the absence of secondary
antibodies which can cause non- specific binding.
High background due to the presence of Secondary
antibodies which may result in non-specific binding.
Ability to stain with multiple antibodies at once since
host factor is not a factor.
Staining with multiple antibodies can be problematic
since host species needs to be considered.
Low sensitivity due to one-step staining method. Higher sensitivity because multiple secondary
antibodies can bind to one primary antibody.
Few direct antibody conjugates are readily available in
the market.
Secondary antibody conjugates are readily available
on the market.
10. Popular Terms
• Immunofluorescence blocking- Blocking is essential for preventing non-specific binding of antibodies or
other reagents to the tissue. To mitigate nonspecific binding, a blocking step should be carried out before
incubation with the primary antibody.
• Cyclic Immunofluorescence (CycIF) - Is a highly multiplexed method for single cell imaging. It allows
fluorescent imaging to different number of proteins/signals that can be simultaneously measured.
• Colocalization Immunofluorescence - Colocalization refers to the observation of the spatial overlap between
two distinct fluorescent markers, each with a different emission wavelength, to see if the different "targets"
are in the same region of the cell or very close to one another in fluorescence microscopy.
11. Popular Terms
• immunofluorescence staining- is an antigen-detection test that is used
primarily on frozen tissue sections, cell “smears,” or cultured cells; formalin-fixed
tissue samples are generally not useful with this procedure.
• Double immunofluorescence- Primary antibodies raised in different species can
be used either in parallel (in a mixture) or in a sequential way to investigate the
co-distribution of two (or more) distinct antigens in the same sample.
• Autofluorescence -This is the normal emission of light by biological structures
such as mitochondria and lysosomes after they have absorbed light, and it is used
to distinguish light from fluorescent markers that have been inserted artificially.
12. Popular Terms
• Multiplex Immunofluorescence- Immunofluorescence technologies that allow multiple
markers to be detected simultaneously on a single tissue segment.
• Double Immunofluorescence staining- This is a staining method of targeting two proteins
within a cell or tissue with two antibodies from two different host species (e.g. mouse IgG
and rabbit IgG), isotypes of the same species.
• FISH immunofluorescence- Fluorescence in situ hybridization (FISH) is way to visualize and
map the genetic material in an individual's cells, including specific or portions of genes.
13. Popular Terms
• Full House immunofluorescence- This simply means that all five main
immunofluorescent stains (IgM, IgG, IgA, C3, and C1q) on a renal
biopsy are positive.
• Linear Immunofluorescence- It is a characteristic pattern of linear
immunofluorescent staining for immunoglobulin G (IgG).
• Multicolor Immunofluorescence- Multicolor immunofluorescence
staining is best carried out by sequentially incubating cells with
unlabeled-primary and labeled-secondary antibodies. However when
options are limited, it may also be performed by simultaneous
incubation of cells with directly labelled primary antibodies.
14. Popular Terms
• Spillover effect - Fluorescence spillover (also
called bleed through or crosstalk) and it can be
encountered in other techniques using
fluorescence such as microscopy.
• Photoluminescence - It is the process through
which atoms emit light following light
absorption, includes fluorescence and
phosphorescence.
15. Commonly used Fluorophores
• Immunofluorescence dyes can be classified as either;
1. Organic dyes
2. Biological Fluorophores
3. Inorganic Fluorophores
•
Characteristics of a good IF Dyes
• Not alter the general shape and function of the target
molecules/cells
• Be localized at the target location on the cell/molecule
• Maintain high specificity even in the presence of other
molecules
• Operate at visible wavelengths
16. Factors That Affect The Efficiency Of
Fluorophores
1. Peak excitation wavelength
2. Peak emission wavelength
3. Quantum yield, brightness
4. Water solubility
5. pH insensitivity
6. Photostability
7. Spillover effect
8.Autofluorescence effects.
17. Organics Dyes
• Fluorescein- Fluorescein is a highly fluorescent substance. It can be detected even when
in very small quantities and is used in microscopy when it's conjugated to antibodies. It is
available as a dark orange / red powder slightly soluble in water and alcohol.
• Multicolor immunofluorescence staining is best carried out by sequentially incubating
cells with unlabeled-primary and labeled-secondary antibodies. However when options
are limited, it may also be performed by simultaneous incubation of cells with directly
labelled primary antibodies. Commonly conjugated to proteins, antibodies, and nucleic
acids for a variety of applications, such as histochemistry, fluorescence microscopy, FISH,
and flow cytometry. The dye has an absorption wavelength that peaks around 495 nm,
and an emission maximum around 517 nm.
• Fluorescein Isothiocyanate (FITC) -Is a derivative of fluorescein and one of the most
widely used organic fluorescent dyes/probes in flow cytometry and immunofluorescence
is fluorescein isothiocyanate. With fluorescein-to-fluorescein interactions resulting in
energy conversion and self-quenching while concentrated, it has a maximum/peak
absorbance of 495nm and an emission wavelength of 520nm.Best reagents for biological
research because of their high absorptivity, excellent fluorescence quantum yield, and
good water solubility.
18. Organics Dyes
• Cyanine Dyes - Cy3 and Cy5 are the most popular, used typically combined for 2
colors detection. Cyanines are resonant dyes characterized by polymethine dyes
between nitrogen atoms (two atoms of nitrogen) with a delocalized charge so that
they can be chemically linked to either nucleic acids or protein molecules. Cyanines
have become one of the most common fluorescent dyes for labeling nucleic acids
due to their low non-specific binding to biomolecules and bright fluorescence.
Because they yield brighter and more stable fluorescence, cyanines can
advantageously replace conventional dyes such as fluorescein and rhodamines.
• Rhodamine- The rhodamines are xanthene derivatives structurally related to
fluorescein, but with additional chemical substitutions that shift their excitation and
emission spectra to longer wavelengths. Rhodamines have superior photostability
and a variety of photophysical properties, making them suitable for use as laser
dyes, fluorescent probes, and pigments as opposed to other dyes on the market
19. Pros and Cons of Organic Dyes
Pros Cons
Photostability A relatively high rate of photobleaching
Most are soluble in Water and Alcohol Fluorescent signal is sensitive to pH changes
Bright Fluorescence A relatively broad fluorescence emission
spectrum
High absorptivity Fluorescence quenching on conjugation to
biopolymer
Excellent fluorescence quantum yield
20. Biological Fluorophores
• Green Fluorescent Protein (GFP) - Green
fluorescent protein (GFP) is a protein in the
jellyfish that exhibits green fluorescence when
exposed to light. The protein has 238 amino
acids, three of them (Numbers 65 to 67) form a
structure that emits visible green fluorescent
light. In the jellyfish, GFP interacts with another
protein, called aequorin, which emits blue light
when added with calcium. GFP is also used to
study cells in embryos and fetuses during
developmental processes.
21. Common Applications of GFP
1. Study of bacterial protein localization
2. Host–pathogen interaction research
3. Use of GFP as reporter gene
4. GFP as active indicator
5. GFP as fusion tag
22. Biological Fluorophores
• Phycoerythrin - PE (phycoerythrin) is a pigment complex made up of red proteins that belongs
to the phycobiliprotein family. It's present in both red algae and cryptophytes, where it serves as
a supplement to the chlorophyll pigments. PE (phycoerythrin) is a pigment complex made up of
red proteins that belongs to the phycobiliprotein family. It’s primary absorption peak is at 565
nm with secondary peaks at 496 and 545 nm. The broad excitation spectrum provides the
advantage for multi-color immunofluorescent staining or cell sorting. They are significantly
brighter and more photostable than conventional organic fluorophores. While it emits bright
fluorescence, phycoerythrin photobleached rather quickly, limiting its use in fluorescence
microscopy.
• Allophycocyanin (APC)- Allophycocyanin, like Phycoe
• rythrin, is derived from red algae and belongs to the phycobiliprotein family. The fluorophore's
absorbance limit is at 650nm, while fluorescence emission peaks at 66nm, when excited by laser
lines at 594 and 633 nm. APC is fluorescent, with an extremely high absorptivity and high
quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by
conventional protein cross-linking techniques without altering its spectral characteristics.
Allophycocyanin is the least stable among the major phycobiliproteins, susceptible to
dissociation at low concentrations including concentrations at which some assays are
performed. APC can be excited by light over a wide range of the visible spectrum, is highly water
soluble, has a relatively low isoelectric point, and lacks potentially sticky carbohydrates
23. Pros and Cons of Biological Fluorophore
Pros Cons
Relatively low isoelectric point Photobleached
Broad excitation spectrum Susceptible to dissociation at low concentrations
Significantly brighter and more photostable Toxicity
Highly Water Soluble Interference with normal biological processes
24. Inorganic Fluorophore
• Quantum Dots - Quantum dots are nanocrystals
(inorganic nanocrystals) with sizes ranging from 2 to 50
nanometers. The light emitted varies in color from blue
to red, depending on the size (with small QT exhibiting
blue light while larger ones exhibit red light). They are
suitable for processes such as immunolabeling,
multiplexed biological detection as well as molecular
imaging both in vitro and in vivo assays. They have much
increased brightness, narrow emission spectrum, large
Stokes shift and photostability compared with
conventional organic fluorescent dyes, which together
make them the fluorophores of choice for demanding
requirements.
25. Inorganic Fluorophore
• Nanoparticles- Nanoparticles can be coated/modified with
fluorophores on their surfaces to make them fluorescent and
improve such properties brightness, biocompatibility and cell-
permeability. Compared with conventional fluorescent dyes,
the use of fluorescent nanoparticles as labels in
immunofluorescence microscopy offers advantages of higher
luminescence and higher photostability. This method can
integrate with epifluorescent filter techniques to further
shorten the time needed for detection. In addition, by
substituting the antibody to suit other bacteria, this technique
has the potential to develop to a universal method for
detecting a wide variety of bacteria in biomedical and
biotechnological areas.
26. Pros And Cons Of Inorganic Fluorophores
Pros Cons
Have much increased brightness, Exhibit in vivo toxicity
Narrow emission spectrum Irregular blinking in case of Quantum dot single cell
tracking.
Large Stokes shift
Higher luminescence and higher photostability
27. Diseases Diagnosed by Immunofluorescence
• Pemphigus vulgaris - Immunofluorescence is one of the diagnostic tests done in
cases of pemphigus. Pemphigus vulgaris is an autoimmune blistering condition
marked by suprabasal blisters.
• Antinuclear Antibody (ANA) - The antinuclear antibody (ANA) test is used as a
primary test to help evaluate a person for autoimmune disorders that affect many
tissues and organs throughout the body (systemic) and is most often used as one of
the tests to help diagnose systemic lupus erythematosus (SLE).
•
Dermatitis Herpetiformis- Granular IgA deposits in dermal papillae of perilesional
skin observed by direct immunofluorescence is the criterion standard of diagnosis.
DH is usually confirmed with a skin biopsy and a specialized type of
immunofluorescent stain that helps to detect the IgA antibodies.
•
Alport syndrome - Immunofluorescence analysis using the monoclonal antibody to
the 28-kilodalton monomers of the non collagenous domain of type IV collagen can
verify the diagnosis of heterozygous Alport syndrome.
•
28. Diseases Diagnosed by Immunofluorescence
Amyloidosis- Amyloidosis is a relatively rare disease characterized by the extracellular deposition of
precursor proteins. Cardiac involvement can be especially devastating due to symptoms of diastolic
dysfunction and right-sided heart failure. Diagnostic algorithm of cardiac amyloidosis of direct
evaluation of the tissue with immunofluorescence and of genetic testing.
Autoimmune Bullous Dermatoses- Immunofluorescence assays plays important role in diagnosing
autoimmune blistering diseases, and higher sensitivity for indirect immunofluorescence when Salt-
split skin technique is performed.
Autoimmune Liver diseases- Autoimmune liver diseases comprising the triad of autoimmune
hepatitis, primary biliary cholangitis (PBC) (cirrhosis) and primary sclerosing cholangitis and their
overlap syndromes are uncommon. . For routine diagnostic immunology laboratories, initial screening
for liver autoantibodies by immunofluorescence remains the method of choice
Chikungunya Virus- Immunofluorescence assay (IFA) is a highly versatile and sensitive assay for
detection and titration of chikungunya virus (CHIKV). The IFA technique requires virus-infected cells
(viral antigen) and antibodies specific to the viral antigens for detection.
•
