Wavelength and Light Properties
575 nm
High
Energy
n=c/l
E = hn
(l)
Low
Energy
Discovery of the Fluorescence: 1852
Sir George Gabriel Stokes
(1819-1903)
Unvisible
UV Light
Fruorite
Visible
Blue Light
Fluorescence
- escence - means "to become" in Latin.
- Fluor comes from the Fluorite (fluorspar)
Fluorescence
Spectral Shift & Filters
( Stokes Shift)
Fluorochrome
(khroma crwma color)
a fluorescent molecule
Fluorophore
(-phore joroz to bear, carrying)
a component of a molecule which causes a molecule to be fluorescent
Fluorescent dye
XXX is labeled with a fluorescent dye …
Fluorescent probe
XXX is the Fluorescent probe for ...
Fluorochromes
Fluorescein isothiocyanate (FITC)
Hoechst
Fluorochromes
Quantum Yield (efficiency)
QY
(QE)
the ratio of the number of photons emitted
to the number of photons absorbed.
Molar extinction coefficient
ME
the ability of a molecule to absorb light
Fluorochromes
Quantum Yield
total integrated photon emission over the entire spectral band of the fluorophore
S1 > S2
Fluorochromes
FITC
ME - 93 000
QY - 0,87
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Phycoerythrin (PE)
ME - 1 960 000
QY - 0,82
Drs E.D. Wieder and L. Gibson, Gladstone
Inst. Of Virology & Immunology, San-Francisco, CA
Fluorescent probes
for DNA
Hoechst
Immunolabeling
Ab ’
Ag … Primary antibody (Ab ’ )-FITC
Ab ’’
Ag … AC ’ … Secondary Antibody ( Ab ’’ )-FITC
Ag … AC ’ -Avidin (a) … Biotin (b)-FITC
a
b
Ag … AC ’ … Protein A (pA)-FITC
pA
Fluorescent Proteins
Ag … Antibody-Fluorochome
Protein … Fluorochome
(Fluorescent Fusion Protein)
Green Fluorescent Protein
Fluorescent probes
for Metal Ions
Fluo-3
Ca++
Fluorochromes
Phycoerythrin
(phycoerithrobilin)
Texas Red
Conjugated Fluorochromes
Texas Red
Phycoerythrin
Red 613
Conjugated Fluorochromes
FL1 FL2 FL3
Laser 488 nm
Quantum dot
Fluorochromes & Cytometer
Cells
(Beads etc)
Fluorochromes
Staining
Instrumentation
“the art and science of measurement”
Data
Sorting
Analysis & Presentation
Examples