1. Fluorescent dyes and their use as biological probes Nico Stuurman Microscopy Course UCSF April 19, 2010

2. Excitation & emission maxima Extinction coefficient  ∝ absorption cross section  ≈ 50,000–100,000 M -1 cm -1 Fluorescence quantum yield Q f = # Photons emitted / # photons absorbed Q f ≈ 25–90% Brightness ∝  Q f Photo-bleaching quantum yield Q b = average # of photons emitted per molecule before bleaching. Depends on environment. ∝ Q f / Q b Parameters of fluorescent dyes I exc l C  : extinction coefficent (M -1 cm -1 ) l : length (cm) C : concentration (M) I in I out I out = I in 10 -  lC

3. Fluorescent dyes/probes Other important factors Lifetime Typically nanoseconds, dye specific Strongly influenced by environment Sensitivity to environment pH, Ca-ions, Coupling Amino- or cystein coupling, number of dyes per molecule, expression as fusion protein Delivery Method Cell permeability, micro-injection, genetic expression, etc.

4. Fluorescent Dye Types Organic dyes Phycobiliproteins Lanthanide Chelates Fluorescent Nanocrystals Fluorescent Proteins

5. Organic Dyes FluoresceinRhodamineCoumarin 554/573 QY 0.28 490/520 QY 0.925 332/456 QY 0.77 The Classics Systems of conjugated bonds that share electrons Larger system → longer wavelength

6. The Enemy: Photo-bleaching Decrease in emission intensity after exposure Exciting a molecule once has a probability Q b of killing it Each molecule will emit only a finite number of photons

7. 0 1 2 S0S0 S1S1 S2S2 hAhA hAhA Internal conversion hEhE Fluorescence T1T1 Phosphorescence hPhP hAhA hAhA O2O2 O 2 Singlet Oxygen Bad! Except when used in CALI (Chromophore-assisted light inactivation) Photo-Bleaching - Mechanisms Zondervan et al., J. Phys. Chem. A, 2004, 108:1657–1665.

8. What to do about photo-bleaching? Select fade-resistant dyes Label densely Decrease bleaching by anti-fade mounting media Glycerol Oxygen scavengers Free-radical scavengers Triplet state quenchers Note: some anti-fade agents quench some dyes. Budget the photons you have Only expose when observing Minimize exposure time & excitation power Use efficient filter combinations Use highly QE, low noise camera Use simple light path

9. Organic Dyes 554/568 QY 0.14 652/672 QY 0.18 Also, Cy2, Cy5.5 Cyanine dyes Alexa dye series 499/517 QY 0.60

10. Organic Dyes Alexa series (a commercial, not chemical) family from Molecular Probes

11. Parameters of some fluorophores Dyel ex l em εQYbrightness DAPI350470 270000.5815.7 Fluorescein490520 670000.7147.6 Alexa 488494517 730000.643.8 Rhodamine554573 850000.2823.8 Cy35545681300000.1418.2 Cy56526722000000.1836 GFP488507 560000.633.6 mCherry587610 720000.2215.8 CFP433475 325000.413 YFP516529 770000.7658.5

12. From Dye to Probe: Small dyes that are Probes DAPI Hoechst33258 Hoechst 33342 ~20 fold enhancement TOTO, YOYO Mitotracker Oxidized in mitochondria in fluorescent compound

13. Conjugation of organic dyes Amino groups (lysine): succinimidyl ester or isothocyanate Small molecules, i.e. phalloidin, taxol Proteins: labeling site unspecific Antibodies: direct/indirect labeling (Label density) Chemistry/Method Targets Example: Dynein driven gliding of microtubules labelled with TMR on lysine residues.

14. Fluorescent labeling Direct labeling (& microinjection) of target molecules Direct immunofluorescence: labeled antibodies against target Indirect immunofluorescence: Unlabeled antibodies against target Labeled antibodies against those antibodies

16. FlAsH/ReAsH Labeling protein with tetra-cysteine motifs (Tsien lab/Invitrogen): Example: Newly synthesised connexins (ReAsH:Red) are added to the outer edges of existing gap junctions (FlAsH:Green). Gaietta et al 2002 Site-Specific Labeling Sulfhydryl groups (cystein): maleimide Chemistry/Method Engineer Cys-light version of target protein

17. HaloTag (Promega) – Dehalogenase SNAP Tag (Covalys) - O6-alkylguanine-DNA alkyltransferase (AGT) Covalent attachment of dyes to genetic tags Your Favourite Protein HaloTag TMR-ligand - labels with anything (biotin/Cy5) - Multiple colors - organic dyes can be brighter - Stoichiometry control - pulse chase possible - labels with anything (biotin/Cy5) - Multiple colors - organic dyes can be brighter - Stoichiometry control - pulse chase possible

18. Non-Covalent Labeling Tris or Tetra-NTA Affinities down to the low pM Peptide sequences evolved to bind fluorophores High-affinity drug-binders FKPB12(F36V) - 12 kDa tag - SLF’ binds at 94 pM

19. Phycobiliproteins Proteins present in cyanobacteria and certain algae Chromophore: covalently bound phycobilins High QY and Absorption coefficient, long lifetime Large -> Antibody conjugate

20. Lanthanide Chelates Chelated rare earth ions Large Stokes shift, long lifetime ChelateExcitation (nm)Emission (nm) Europium (Eu)340615 Samarium (Sm)340642 Terbium (Tb)300545

21. Quantum-dots Advantages Very bright Very photostable Excitation possible at a single wavelength Visible in electron microscope nanometre-scale crystals composed of atoms of an inorganic semiconductor material Disadvantages Large size Multivalent linkage

22. Quantum dot labeling Biotin/Streptavidin Linkage Biotin maleimide (in vitro) Biotin HaloTag/SNAP Biotin carrier protein BiotinLigase/AP1 Antibody Conjugates – immunohistochemistry Direct linkage to proteins/peptides – targeting to cell compartments Qdot labelled dynein via HaloTag:Biotin:Streptavidin linkage moving on axonemes

23. Fluorescent proteins Discovery Images from Osamu Shimomura

24. No co-factors needed! First GFP expression in E. coli and C. elegans Images from Martin Chalfie Douglash Prasher

25. The green fluorescent protein Day and Davidson, Chem Soc Rev, 2009(38) 2887 ~240 Amino acids, 27 kD GFP

26. Improving the wild type GFP Day and Davidson, Chem Soc Rev, 2009(38) 2887 AvGFP“GFP” S65T

27. GFP-Actin in Drososophila S2 cells Steve Rogers, 2002

28. Jellyfish (Aequorea) fluorescent protein family AvGFP ↓ GFP (S65T), EGFP (S65T, F64L) ↓ BFP, CFP, YFP ↓ Cerulean, CyPet, Sapphire, Venus, Citrine, Ypet… AvGFP ↓ GFP (S65T), EGFP (S65T, F64L) ↓ BFP, CFP, YFP ↓ Cerulean, CyPet, Sapphire, Venus, Citrine, Ypet…

29. Seeking red colors… Lukyanov lab: PCR with degenerate primers on tropical corals (from Moskow pet shop)

31. Oligomerization Monomer / weak dimer Tetramer GFPdsRed

32. Fluorescent proteins Protein  ex  em  QYBrightnessSource CFP433475325000.413.0Tsien GFP488507560000.633.6Tsien Citrine516529770000.7658.5Tsien PhiYFP5255371300000.452.0Evrogen MkOrange548559516000.631.0Miyawaki tdimer25525791200000.6881.6Tsien tdtomato5545811380000.6995.2Tsien DsRed-monomer556586Clontech mRFP1584607440000.2511.0Tsien mCherry587610720000.2215.8Tsien tHcRed5906371600000.046.4Clontech

33. Switchable fluorescent proteins Color-changing Green-red: Kaede, EosFP, KikGR,… Cyan-green: PS-CFP Fluorescence that can be activated or altered by light PA-GFP (T203H), 100:1 contrast George H. Patterson and Jennifer Lippincott-Schwartz, 2002 Activatable PA-GFP, … Reversibly switchable KFP, Dronpa

34. Acknowledgements and Resources Kurt Thorn Bo Huang Mats Gustaffson Andrew Carter Lakowicz - Principles of Fluorescence Spectroscopy Goldman et al. - Live Cell Imaging: A Laboratory Manual Day and Davidson, Chem Soc Rev, 2009(38) 2887