1. Principles of fluorescent probes Anna Dubiel Warsaw, 17.05.2011

2. Plan of presentation -> introduction in fluorescence microscopy -> basics of fluorescence -> properties of fluorescent probes -> cell probing: > DNA > membranes > tubulin > mitochondria > indicators for Ca 2+ > pH -> summary 2

3. Disadvantages of optical microscopy -Usual microscopes working at visible spectral range have magnification coefficient up to 1 500x -Microscopes for ultra-violet have magnification coefficient up to 3500x -Majority of cell components are transparent and colourless -Dyes used in traditional microscopy often are not selective and paint whole cell 3

4. Advantages of fluorescent microscopy -Higher sensitivity -Using visible or near IR spectral range -Dyes which are specific for subcellular components, proteins or ions -Observation of cell division -3D 4

5. Jabłoński diagram Basics of fluorescence Aleksander Jabłoński 1898 – 1980 Profesor of Nicolaus Сoperniсus University in Torun IC + VR 5

6. Basics of fluorescence Absorption: Fluorescent quantum yield: Jabłoński diagram Brightness: Stokes shift: IC + VR 6

7. Fluorescence microscope : 7

8. Dyes: - High absorption - High fluorescent quantum yield - Water solubility - Affinity to a particular part of the cell - Chemical and photostability - Stability in cell conditions - Low cytotoxicity 8

9. Dyes for imaging: Rhodamine dyesFluoresceines fluoresceine λ abs =489nm λ em =534nm ϕ f =0,73 ε = 92 300 M -1 cm -1 J. Mater. Chem., 2009, 19, 2018–2025 rhodamine B λ abs =542nm λ em =579nm ϕ f =0,50 ε = 106 000 M -1 cm -1 9

10. Dyes for imaging : Cyanines Angew. Chem. Int. Ed. 2009, 48, 299 –303 Chem. Phys. Lett., 1978, 54, 159-163 Cy3 λ abs =546nm λ em =571nm ϕ f =0,05 ε = 271 000M -1 cm -1 Coumarins coumarin 440 λ abs =354nm λ em =434nm ϕ f =0,73 ε = 23 500M -1 cm -1 Chem. Med. Chem. 2010, 5, 103 – 117 10

11. Dyes for imaging : BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene λ abs=499nm λ em=535nm ϕ f =0,93 ε > 80 000 M -1 cm -1 J. Org. Chem. 2009, 74, 5719– 5722 11

12. DNA detection DAPI Λ abs=358nm λ em=461nm ϕ f =20% The Molecular Probes Handbook; A Guide to fluorescent Probes and Labeling technologies; Eleventh Edition Ewa M. Goldys, Fluorescence Aplications in Biotechnology and the Life Science; Wiley-Blackwell; 2009 Cy5-dUTP λ abs=649nm λ em=670nm ϕ f =28% 12

13. Probes for membranes ß-BODIPY FL C 5 -HPA λ abs =504nm λ em =511nm ε= 79 000 M -1 cm -1 Fluorescein DHPE λ abs =496nm λ em =519nm ε= 88 000 M -1 cm -1 13

14. Probes for tubulin BODIPY FL vinblastine λ abs =503nm λ em =510nm ε= 83 000 M -1 cm -1 Oregon Green 488 Taxol λ abs =494nm λ em =522nm ε= 80 000 M -1 cm -1 14

15. Probes for mitochondia MitoTracker Red CMXRos λ abs=578nm λ em=599nm ε = 116 000 M -1 cm -1 MitoTracker Green FM λ abs=490nm λ em=516nm ε = 119 000 M -1 cm -1 15

16. Indicators for Ca 2+ Calcium Orange λ abs=549nm λ em=575nm ε = 80 000 M -1 cm -1 Fura Red λ abs=473, 436nm λ em=670, 655nm ε = 29 000, 41 000 M -1 cm -1 16

17. pH indicators 17 5-(and-6)-carboxy SNARF-1 λ abs=548, 576nm λ em=587, 635nm ε = 27 000, 48 000 M -1 cm -1

18. Summary Fluorescence microscopy: -> An essential tool in biology and the biomedical sciences -> Based on fluorescence fenomena -> Use fluorescent probes Aplication of fluorescence microscopy: 1.Detection and determination of the proteins localization in cell and tissue 2.Examination changes of ions concetration 3.Diagnostic of diseases 18

19. Thank you for your attention 19