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

2. Sir George Gabriel Stokes (1819-1903) Unvisible UV Light Visible Blue Light Fluorescence Fruorite Discovery of the Fluorescence: 1852 - escence - means "to become" in Latin. - Fluor - comes from the Fluorite (fluorspar)

3. Fluorescence

4. Spectral Shift & Filters ( Stokes Shift )

5. 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...

6. Fluorescein isothiocyanate (FITC) Hoechst Fluorochromes

7. 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.

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

9. Drs E.D. Wieder and L. Gibson, Gladstone Inst. Of Virology & Immunology, San-Francisco, CA Fluorochromes FITC ME - 93 000 QY - 0,87 Phycoerythrin (PE) ME - 1 960 000 QY - 0,82

10. Hoechst Fluorescent probes for DNA

11. 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

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

13. Fluo-3 Ca ++ Fluorescent probes for Metal Ions

14. Phycoerythrin (phycoerithrobilin) Texas Red Fluorochromes

15. Phycoerythrin Texas Red Red 613 Conjugated Fluorochromes

16. FL1FL2FL3 Laser 488 nm Conjugated Fluorochromes

17. Quantum dot

18. Fluorochromes & Cytometer

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