History

In 1941, Albert Coons

demonstrated that
antibodies could be
labeled with molecules
that fluoresce.

These fluorescent
compounds are called
fluorophores or
fluorochromes.
Dr. Albert Coons
 Fluorochromes
Fluorophores are typically
organic molecules with a
ring structure, and each
has a characteristic
optimum absorption
range.

The edge of a tumor where the levels of

different proteins has been probed for using
antibodies labelled with fluorophores.
 Principle
Fluorophores can absorb
energy (excitation energy)
from an incident light
source and convert that
energy into light of a longer
wavelength and lower
energy as the excited
electrons return to the Transformed African Green Monkey
ground state. Kidney Fibroblast Cells (COS-7 Line)
 Principle
The time interval
between absorption of
energy and emission of
fluorescence is very short
and can be measured in
nanoseconds (ns).
 Principle
Ideally, a fluorophore or a fluorescent probe
should exhibit a high intensity and stability

Incident Light Fluorescence

Ground State Electrons go to highest Electrons return to

orbitals (excitation) lowest orbitals
(ground state)
 Principle
The difference
between the
wavelength of the
excitation light
and the emission
light is called the
Stokes shift.
 Immunofluorescence
Fluorescence immunoassay is a
sensitive technique that can be
used in the measurement of
many compounds, including
drugs, hormones, and proteins;
in the identification of
antibodies; and in the
quantification of antigens such
as viral particles and, Epifluorescent micrographs of frozen sections
of a normal rabbit retina as seen through a
potentially, bacteria. fluorescein isothiocyanate detection filter (left
column) and a rhodamine B isothiocyanate
detection filter (right column).
Typical Fluorophores
Fluorescein isothiocyanate (FITC) Most often used;
can be readily
coupled with
Tetramethylrhodamine isothiocyanate (TRITC) antigen or antibody

Phycoerythrin

Europium (ß – naphthyl trifluoacetatone)

Lucifer Yellow VS
Fluorophores

Fluorescein isothiocyanate (FITC) Tetramethylrhodamine

isothiocyanate (TRITC)

GREEN FLUORESCENCE RED FLUORESCENCE

Green emission @517-585nm Red emission @580-585
 Immunofluorescence
Fluorescent tags or labels were first
used for histochemical localization of
antigen in tissues.

This technique is called

immunofluorescent assay (IFA),
qualitative observations involving the
use of a fluorescence microscope.

Many types of antigens can be

detected either in fixed tissue sections
or in live cell suspensions with a high
degree of sensitivity and specificity.
 Immunofluorescence
The presence of a specific antigen is
determined by the appearance of
localized color against a dark
background.

This method is used for rapid

identification of microorganisms in cell
culture or infected tissue, tumor-
specific antigens on neoplastic tissue,
transplantation antigens, and CD
antigens on T and B cells (flow
cytometry).
References
• Stevens, Christine Dorresteyn. (2010) Clinical Immunology & Serology A Laboratory
• Perspective. Agglutination. p. 158. Philadelphia, PA. : F.A. Davis.
• https://www.leica-microsystems.com/science-lab/explore-innovative-techniques-to-separate-
fluorophores-with-overlapping-spectra/
• https://www.quidel.com/immunoassays/fluorescent-immunoassays-fia
• https://pubmed.ncbi.nlm.nih.gov/6365732/