Wavelength and Light Properties 575 nm  c  E = h  High Energy Low Energy

Sir George Gabriel Stokes ( ) Unvisible UV Light Visible Blue Light Fluorescence Fruorite Discovery of the Fluorescence: escence - means "to become" in Latin. - Fluor - comes from the Fluorite (fluorspar)

Fluorescence

Spectral Shift & Filters ( Stokes Shift )

Fluorochrome (khroma  color) a fluorescent molecule Fluorophore (-phore  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...

Fluorescein isothiocyanate (FITC) Hoechst Fluorochromes

Molar extinction coefficient ME the ability of a molecule to absorb light Quantum Yield (efficiency) QY ( QE) the ratio of the number of photons emitted to the number of photons absorbed.

Fluorochromes Quantum Yield total integrated photon emission over the entire spectral band of the fluorophore S1 > S2

Drs E.D. Wieder and L. Gibson, Gladstone Inst. Of Virology & Immunology, San-Francisco, CA Fluorochromes FITC ME QY - 0,87 Phycoerythrin (PE) ME QY - 0,82

Hoechst Fluorescent probes for DNA

Ag … AC ’ -Avidin (a) … Biotin (b)-FITC a b Ag … Primary antibody (Ab ’ )-FITC Ag … AC ’ … Protein A (pA)-FITC Ag … AC ’ … Secondary Antibody ( Ab ’’ )-FITC pA Ab ’ Ab ’’ Immunolabeling

Green Fluorescent Protein Fluorescent Proteins Ag … Antibody-Fluorochome Protein … Fluorochome (Fluorescent Fusion Protein)

Fluo-3 Ca ++ Fluorescent probes for Metal Ions

Phycoerythrin (phycoerithrobilin) Texas Red Fluorochromes

Phycoerythrin Texas Red Red 613 Conjugated Fluorochromes

FL1FL2FL3 Laser 488 nm Conjugated Fluorochromes

Quantum dot

Fluorochromes & Cytometer

Instrumentation “the art and science of measurement” Data Analysis & Presentation Sorting Cells (Beads etc) Fluorochromes Examples Staining