Immunofluorescence is a technique developed in 1944 that labels antibodies with fluorescent dyes to visualize antigens under a fluorescence microscope. The process employs light of specific wavelengths to excite fluorochromes like fluorescein and rhodamine, which emit light of longer wavelengths. Applications include direct and indirect immunofluorescence for disease diagnosis and flow cytometry (FACS) for cell sorting and analysis.

1. IMMUNOFLUORESCENCE
DR. SHILPY SINGH
ASSTT. PROFESSOR
MIMT, GR. NOIDA

2. Introduction:
 In 1944, Albert Coons showed that antibodies could be labelled with
molecules that have a property of fluorescence.
 Fluorescent molecules absorb light of one wavelength and emit light of
another wavelength.
 Immunofluorescence is the labeling of antibodies or antigens with
fluorescent dyes, or fluorochrome.
 This technique is sometimes used to make viral plaques more readily visible
to the human eye.
 Immunofluorescent labeled tissue sections are studied using a fluorescence
microscope i.e the epifluorescence microscope, and the confocal
microscope
 Fluorescein is a dye which emits greenish fluorescence under UV light. It
can be tagged to immunoglobulin molecules.

3. The basic principle of
immunofluorescence
 To use a fluorescent compound (usually
fluorescein and rhodamine) to detect the binding
of antigen and antibody
 The Ab is labelled with the fluorescent
compound
 Under a fluorescence microscope, fluorescein
appears bright green and rhodamine appears
orange/red wherever the binding occurs

4. Technique:
 Common dyes: fluorescein isothiocyanate (FITC) or tetramethyl
rhodamine isothiocyanate (TRITC)
 Dyes chosen are excited by a certain light wavelength, usually blue
or green, and emit light of a different wavelength in the visible
spectrum
 Eg. Fluorescein emits green light
 Eg. Rhodamine emits orange/red light
 Highly fluorescent substances such as phycoerythrin and
phycobiliprotein have also been used.
 By using selective filters in a fluorescence microscope only the light
from the dye is detected
 Available fluorescent labels now include red, blue, cyan or yellow
fluorescent proteins

5. STOKES FLUORESCENCE:
 The phenomenon of fluorescence was first
explained by a British scientist, Sir George
Stokes, in 1852, the shift in wavelength from
short to long during fluorescence is called
“Stokes shift”
 Stokes fluorescence is the re-emission of
longer wavelength photons by a molecule that
has absorbed photons of shorter wavelengths.
 Both absorption and emission of energy are
unique characteristics of a particular molecular
structure. If a material has a direct band gap in
the range of visible light, the light shining on it
is absorbed, causing electrons to become
excited to a higher energy state.
 The electrons remain in the excited state for
about 10-8 seconds. This number varies over
several orders of magnitude, depending on
the sample and is known as the fluorescence
lifetime of the sample. The electron returns to
the ground state and energy is emitted.

6. FLUORESCENT MICROSCOPE
 Several microscope designs can
be used for analysis of
immunofluorescence samples.
 The specimen is illuminated with light
of a specific wavelength which is
absorbed by the fluorophores,
causing them to emit light of longer
wavelengths
 The illumination light is separated
from the much weaker emitted
fluorescence through the use of a
spectral emission filter.

7. Components of a fluorescence
microscope
 Light source (xenon
arc lamp or mercury-
vapor lamp)
 Excitation filter
 Dichroic mirror (or
dichromatic
beamsplitter), and
 Emission filter

8.  APPLICATIONS OF IMMUNOFLUORESCENCE:
Direct immunofluorescence
Indirect immunofluorescence
FACS (Fluorescence activating cell sorting)

9. Direct immunofluorescence
Uses:
Direct detection of Pathogens or
their Ag’s in tissues or in pathological
samples
Also used for localization of IgG in
immune complexes along the dermal-
epidermal junction of skin biopsies
from patients suffering from systemic
lupus erythematosus
The aim is to identify the presence and
location of an antigen by the use of a
fluorescent labeled specific antibody

10. Advantages of direct immunofluorescence:
 Shorter sample staining times and simpler dual and triple labeling
procedures.
 In cases where one has multiple antibodies raised in the same
species, for example two mouse monoclonal, a direct labeling may
be necessary.
Disadvantages of direct immunofluorescence:
 Lower signal, generally higher cost, less flexibility and difficulties
with the labeling procedure when commercially labeled direct
conjugates are unavailable

11. Indirect immunofluorescence
 The aim is to identify the presence of
antigen specific antibodies in serum. The
method is also be used to compare
concentration of the antibodies in sera.
 Indirect test is a double-layer technique,
uses two antibodies i.e the primary
antibody and secondary antibody, which
carries the fluorochrome
 The most widely used method of IF in
pathology.
USES:
 For the diagnosis of bacterial, viral and
protozoan diseases including:
 Borrelia burgdorferi ,
 Rickettsia rickettsiae, Rocky Mountain
Spotted Fever,
 Bovine immunodeficiency like virus and
 Toxoplasma gonadii

12.  Advantage over direct IF
 The primary antibody does not need to be conjugated
with a fluorochrome because the supply of primary
antibody is often a limiting factor, indirect methods
avoid the loss of antibody that usually occurs during
the conjugation reaction.
 Indirect methods increase the sensitivity of staining
because multiple molecules of the fluorescence
reagent bind to each primary antibody molecules,
increasing the amount of light emitted at the location of
each primary antibody molecule.

13. FACS (Fluorescence activated cell sorting)
 Fluorescent antibody techniques are extremely valuable qualitative tools,but
do not provide quantitative data, this was remedied by the development of
flow cytometry.
 FACS was used to automate the analysis and separation of cells stained
with fluorescent antibody.
 The FACS uses a laser beam and light detector to count single intact cells
in suspension.
 Cells having a fluorescently tagged antibody bound to their cell surface
antigen are exited by the laser and emit light, an attached computer
generate plots of a no. of cells and their fluorescence intensity.
 Use of the instrument to determine which and how many members of cell
population bind fluorescently labeled antibodies called ANALYSIS.
 Use of the instrument to place cells having different pattern of reactivity in
different containers is CELL SORTING.

14. Flow cytometry
FACS
Fluorescence-
activated cell sorter

15.  FACS now allow the use of multiple fluorescent antibodies.
 Highly sophisticated flow cytometers simultaneously analyze cell
populations that have been labeled with two or even three different
fluorescent antibodies.
 For e.g if blood sample react with a fluorescein tagged antibody
specific for T-cell, and also with phycoerythrin-tagged antibody
specific for B-cell, the percentages of B and T cell may be determine
simultaneously with a single analysis…

16. Uses of FACS
 FACS has multiple uses in clinical and research problems i.e. to
determine the kind and the no. of white blood cells in each
population in patients blood sample, by treating appropriately
processed blood samples with a fluorescently labeled antibody and
performing FACS analysis.
 Also used for the detection and classification of leukemia
depends heavily on the cell types involved.
 FACS also used for the rapid measurement of T-cell sub-
populations, an important prognostic indicator in AIDS. In this
procedure, labeled monoclonal antibodies against the major T-cell
subtypes bearing the CD4 and CD8 antigens are used to determine
their ratio in patients blood. When the number of CD4 T cells falls
below a certain level, a patient is at high risk of opportunistic
infections.

17. LIMITATIONS OF IMMUNOFLUORESCENCE
 PHOTOBLEACHING:
 Photochemical destruction of a fluorophores due to the generation of reactive
oxygen species in the specimen as a byproduct of fluorescence excitation.
 Can be controlled by
(i) reducing the intensity or time-span of light exposure
(ii) increasing the concentration of fluorophores, or by employing fluorophores
that are less prone to bleaching e.g., Alexa Fluors
 AUTOFLUORESCENCE
 Only limited to fixed (i.e., dead) cells when structures within the cell are
to be visualized because antibodies cannot cross the cell membrane.
An alternative approach is using recombinant proteins containing
fluorescent protein domains, e.g., green fluorescent protein
(GFP),these proteins allows determination of their localization in live
cells.

18. Epifluorescent imaging of the three
components in a dividing human
cancer cell.
Endothelial cells under the microscope
Yeast cell membrane visualized by
some membrane protein fused with
RFP and GFP fluorescent markers.

19. THANKS