1. Johan Hofkens Laboratory of Photochemistry and Spectroscopy Katholieke Universiteit Leuven - Belgium K.U.LEUVEN Theories and methods to study molecular interactions : fluorescence and it’s applications

2. 30/11/2005 : Basic principles of fluorescence - absorption, emission, charateristics of a probe - time resolved measurements - quenching, anisotropy - energy transfer, electron transfer - examples 02/12/2005 : Fluorescence microscopy (Dr J. Hotta) - definitions, parameters - different types of microscopy 07/12/2005 : Single molecule fluorescence microscopy - why single molecule studies - different single molecule approaches 09/12/2005 : Applications of fluorescence microscopy Principles of fluorescence and it’s applications to study molecular interactions

3. Fluorescence What is it? Where does it come from? Parameters, Advantages, Techniques Examples

4. http://www.chem.kuleuven.ac.be/research/mds/bioinformatics_courses.htm References & additional reading

6. For light to be useful to us it must interact with matter Types of interaction: –Reflection –Refraction –Absorption (followed by emission) Fluorescence : photons emitted by organic molecules after interaction with light

7. Dual Nature of light: wave and particle –Light as a wave: = c/ E = h = hc/

8. Dual Nature of light: wave and particle –Light as a particle:

9. Visible light –Why do we call this “visible” light Wavelength Range (nanometers) Perceived Color 340-400 Near Ultraviolet (UV; Invisible) 400-430Violet 430-500Blue 500-560Green 560-620Yellow to Orange 620-700Orange to Red Over 700Near Infrared (IR; Invisible)

10. Overview of electromagnetic radiation

12. Absorption : electronic transition(s) in a molecule Orbitals, molecular orbitals

13. Simplified Jablonski Diagram S0S0 S’ 1 Energy S1S1 hv ex hv em

14.  Return to ground state results in emission of radiation (fluorochrome).  Absorption of photon elevates chromophore to excited state.

15. Absorption : Franck Condon Principal, Vibrational fine structure

17. - PES displaced, for molecules where the excitation is delocalized. - Transition from S 0 crosses S 1 with highest probability in 1th vibronic level. - The 0-0 transition is not the most intense one anymore. - This results in a more symmetric spectrum. Characteristics of stationary molecular fluorescence

18. - Effect on emission is similar as for absorption - For rigid molecules with little displacement between PES mirror symmetry and large overlap

19. Characteristics of stationary molecular fluorescence - Effect on emission is similar as for absorption - For rigid molecules with displacement between PES mirror symmetry and small overlap

20. Characteristics of stationary molecular fluorescence - Effect on emission is similar as for absorption - For rigid molecules with displacement between PES mirror symmetry and small overlap

21. Characteristics of stationary molecular fluorescence - Repulsive S 1 PES results in a broad unstructured spectrum. - Maximum given by the AB line. - Symmetric (Gaussian) absorption band.

22. Characteristics of stationary molecular fluorescence - Repulsive ground state, emission will result in a broad band - When stabilizing excited state interaction is caused by two identical molecules it is called excimer, when the interaction is caused by two different molecules it is called exciplex.

26. Stokes shift –is the energy difference between the lowest energy peak of absorbence and the highest energy of emission 495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescnece Intensity Wavelength result of : vibrational relaxation solvent reorganization

27. Stokes shift

28. Fluorophores/chromophores/probes Chromophores are compounds or molecules which absorb light They contain generally aromatic rings The longer the conjugated system, the longer wavelength of fluorescence.

29. Fluorophores/chromophores/probes

31. Allophycocyanin (APC) Protein 632.5 nm (HeNe ) Excitation Emisson 300 nm 400 nm 500 nm 600 nm 700 nm

32. Excitation - Emission Peaks Fluorophore EX peak EM peak % Max Excitation at 488568 647 nm FITC4965188700 Bodipy5035115811 Tetra-M-Rho55457610610 L-Rhodamine5725905920 Texas Red5926103451 CY564966611198

33. Probes for Proteins FITC488525 PE488575 APC630650 PerCP ™ 488680 Cascade Blue360450 Coumerin-phalloidin350450 Texas Red ™ 610630 Tetramethylrhodamine-amines 550575 CY3 (indotrimethinecyanines) 540575 CY5 (indopentamethinecyanines) 640670 ProbeExcitationEmission

34. Hoechst 33342 (AT rich) (uv)346460 DAPI (uv)359461 POPO-1 434456 YOYO-1 491509 Acridine Orange (RNA)460650 Acridine Orange (DNA)502536 Thiazole Orange (vis)509525 TOTO-1514533 Ethidium Bromide526604 PI (uv/vis)536620 7-Aminoactinomycin D (7AAD)555655 Probes for Nucleic Acids

35. DNA Probes AO –Metachromatic dye concentration dependent emission double stranded NA - Green single stranded NA - Red AT/GC binding dyes –AT rich: DAPI, Hoechst, quinacrine –GC rich: antibiotics bleomycin, chromamycin A 3, mithramycin, olivomycin, rhodamine 800

36. Probes for Ions INDO-1 E x 350E m 405/480 QUIN-2E x 350E m 490 Fluo-3 E x 488E m 525 Fura -2E x 330/360E m 510

37. pH Sensitive Indicators SNARF-1488575 BCECF488525/620 440/488525 [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein] ProbeExcitationEmission

38. Probes for Oxidation States DCFH-DA(H 2 O 2 )488525 HE(O 2 - )488590 DHR 123(H 2 O 2 )488525 Probe Oxidant ExcitationEmission DCFH-DA- dichlorofluorescin diacetate HE- hydroethidine DHR-123- dihydrorhodamine 123

39. Specific Organelle Probes BODIPY Golgi505511 NBD Golgi488525 DPH Lipid350420 TMA-DPH Lipid350420 Rhodamine 123 Mitochondria 488525 DiOLipid488500 diI-Cn-(5)Lipid550565 diO-Cn-(3)Lipid488500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium

40. Other Probes of Interest GFP - Green Fluorescent Protein –GFP is from the chemiluminescent jellyfish Aequorea victoria –excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm –contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence –Major application is as a reporter gene for assay of promoter activity –requires no added substrates

41. Excited State Dynamics of the Green Fluorescent Proteins Wild-type GFP HO N N O N OH NH N H 2 N NH 3 + 2 O O HO Serine65 Arginine96 Glutamine94 Tyrosine66 Glu222 Glycine67 Phenylalanine64 Histidine148 Other Probes of Interest

44. Excited State Dynamics of the Green Fluorescent Proteins monitoring proteins, organelles, cells in living tissue. protein-protein interaction using double labeling and FRET. membrane traffic studies. pH sensor. Ca 2+ sensor. ………. Applications : Other Probes of Interest

45. Fluorescent proteins

46. DsRed – a longer wavelength substitute for GFPs

50. New trends in GFP-research Optical marking (following intracellular dynamics) or kindling Patterson, G. H. & Lippincott-Schwartz, J. Science 2002, 297, 1873.

51. Photo-Switchable Fluorescent Protein Dronpa Dronpa is a monomeric GFP-like fluorescent protein from coral Echinophyllia sp. Dronpa shows reversible photoswitching on irradiation with a 488 nm and 405 nm light. On Off Intensity 488 nm 405 nm Time

52. Steady-State Spectra of Dronpa pH = 7.4pH = 5.0 Deprotonated form (B form); fluorescent state,  fl 488 = 0.85,  fl = 3.6 ns Protonated form (A 1 form); dim state,  fl 390 = 0.02,  fl = 14 ps

53. Photoswitching of Dronpa at the Ensemble Level 488 nm 405 nm pH = 7.4

54. Photoswitched Protonated (A 2 ) Form 488 nm 405 nm pH = 5.0

55. Scheme of the Photoswitching On Off Intensity 488 nm 405 nm Time Photoswitched protonated form Non-fluorescent intermediate S0S0 S1S1 Fluorescent deprotonated form  = 3.2 ×10 -4  = 0.37 3.6 ns Protonated form 14 ps

56. New trends in GFP-research Diffraction-unlimited microscopy in far field Hell, S. W. Curr. Opin. Neurobiol. 2004, 14, 599.

57. New probes for fluorescence

59. Emission versus excitation spectrum - Emission spectrum or fluorescence spectrum: one excites at one wavelength and scan the emission- monochromator. - Excitation spectrum : one fixes the emission monochromator at one wavelength and scans the excitation monochromator. - At low concentrations excitation spectra and emission spectra should be the same. Differences point to aggregation or other processes (see energy tranfer).

62. Excitation Sources Lamps Xenon Xenon/Mercury Lasers Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) Krypton-Argon (Kr-Ar)

63. Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 )         Xe Lamp Hg Lamp

64. Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Common Laser Lines

65. Definitions for fluorescence

66. Characteristic times Absorption : 10 -15 s Vibrational relaxation : 10 -12 10 -10 s Lifetime of S1 : 10 -10 10 -7 s Intersystem crossing : 10 -10 10 -8 s Internal conversion : 10 -11 -10 -9 s Lifetime T1 : 10 -6 – 1 s

67. Extinction Coefficient –  refers to a single wavelength (usually the absorption maximum) Quantum Yield –Q f is a measure of the integrated photon emission over the fluorophore spectral band Parameters quantum yield  fl =k / (k fl +k ISC +k IC +k BM ) radiativelifetime  0 = 1/ k fl decay time  fl = 1/ (k fl +k ISC +k IC +k BM ) Lifetime & decay time

68. Parameters Transition dipole moment : direction of movement of electrons

69. Photobleaching Defined as the irreversible destruction of an excited fluorophore (discussed in later lecture) Methods for countering photobleaching (see microscopy) –Scan for shorter times –Use high magnification, high NA objective –Use wide emission filters –Reduce excitation intensity –Use “antifade” reagents (not compatible with viable cells)

70. anisotropy r = (I II - I  )/ (I II +2I  ) polarisationP = (I II - I  )/ (I II +I  ) Definitions for fluorescence Principle of photoselection : using polarized excitation light mainly molecules excited that have a transition dipole parallel to the excitation light. As a result, the fluorescence is also polarized, unless processes occur that ‘destroy’ the polarization Processes can be : rotation of the molecule, energy transfer… Relation between P and r = In ensemble measurements r is most frequently used. In absence of depolarization processes the fundamental of limiting anisotropy value r 0 has a value between 0.4 and -0.2 depending on the angle between excitation and emission transition dipole.

71. Decay time of a fluorophore

72. SAMPLE Excitation (  pulse) d[ 1 M*]/dt = - (k fl +k ISC +k IC +k Q [Q]) [ 1 M*]  fl = 1/(k fl +k IC + k ISC +k Q [Q]) [ 1 M*] = [ 1 M*] 0 exp(-t/  fl ) Fluo. response function I fl (t)  (1/  fl )exp(-t/  fl ) Solving the differential equation Time resolved fluorescence : excitation of the sample with a pulse that is shorter then the decay time of the fluorophore, typically 5 ns. Time resolved fluorecence

73. Basic principle of the TCSPC experiment

74. CFD TAC Excitation source: - flash lamps + monochromatic filters (ns pulses up to 10 kHz rep.) - mode-locked lasers (ps pulses up to 82 MHz rep.) - pulsed semiconductor diode lasers - synchrotron radiation (UV excitation) Optical components: - polarization accessories - collection lens system - monochromator Detection system: - PMT, MCP or APD Electronics: - Delay, CFD, TAC, Amplifier, MCA, PC.

75. Statistics start xixi = q P x (i) = (1/x!)[( ) x exp(- )]  = q i titi  pulse S<ni><ni>MCP Large number of pulses for one event Single Photon Counting ! Decay histogram Y i = (1/  fl )  S exc T  t exp(-i  t/  fl ) Fit functions for the decays : i T (t)=  A j exp(-t/  j ); exponential model i T (t) = Aexp(-t/  fl -2  (t/  fl ) 1/2 ); nonexponential model

76. TCSPC experiment at K.U.Leuven Measurements under ‘magic angle’ in order to avoid distortions by rotational diffusion (magic angle is 54.7 degrees for vertical polarization). polarizer

77. Time-resolved emission spectroscopy (TRES) provide information on the evolution of kinetics in terms of intensity, time and spectral position solvent relaxation around fluorophores, short-lived species, molecules having two or more fluorescing configurations with different decay times are processes that can be studied using TRES.

78. Time-resolved fluorescence depolarization measurements information about the molecular reorientational motion in solution. r (t)= (I II (t) - I  (t))/ (I II (t) +2I  (t)) I T (t) = I II (t) +2I  (t) I II (t)=exp(- t/  fl )(1+2r 0 exp(- t/  )) I  (t)=exp(- t/  fl )(1-r 0 exp(- t/  )) r = r 0 exp(- t/  ) 1.  fl <  r : fluorescence decays before anisotropy  only r 0 can be measured 2.  fl >  r or  fl   r : r 0 and  r can be measured.

79. intra and intermolecular excited state processes taking place from picosecond to nanosecond time scale. determination of rates of competitive de-excitation pathways. reaction kinetics: proton/electron and energy transfer, excimer or exciplex formation. environmental effects: solvent relaxation, quenching of excited states, conformational dynamics in proteins.

80. Energy Transfer Radiative Non radiative -Dexter type - Forster type

81. Energy transfer - Energy transfer is iso-energetic, followed by fast vibrational relaxation - Excited state of acceptor should be lower than that of donor to have driving force - Quantum yield of donor and decay time of donor decrease. - Process can occur between singulet as well as triplet excited states. - Two mechanisms (except for trivial mechanisms) : Dexter and Förster transfer

82. Energy transfer - Dexter transfer : exchange mechanism, distances between 0.5 and 1 nm, spin changes are allowed. Overlap between donor fluorescence and acceptor absorption required.

83. Energy transfer - Förster transfer : long distance, upto 10 nm, dipole-dipole interaction, total spin maintained, resonance energy transfer. Overlap between donor fluorescence and acceptor absorption required. Due to strong distance dependence also called ‘molecular ruler’. - Förster transfer between identical chromophores is called energy hopping and can go in both directions. E is called the efficiency of energy transfer

84. Fluorescence Fluorescene (Forster) Resonance Energy Transfer FRET Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2

85. Energy transfer E can be obtained from the fluorescence quantum yield in the presence (Q DA ) and absence of the acceptor (Q D ) (and in a similar way from decay time in presence and absence of acceptor). It can be shown that the rate constant for transfer equals:  D is the decay time of the donor in absence of the acceptor, R is the distance between donor and acceptor and R 0 is the Förster radius, the distance at witch half of the excitation energy undergoes transfer while half is dissipated by all the other processes including emission. J is the so called overlap integral between emission and absorption and  is the orientation factor (2/3 for random orientation).

86. Energy transfer The overlap integral can be calculated as : The orientation factor can be written as:

89. Energy transfer

90. Forster type Energy Transfer (FRET) Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor PE-Texas Red ™ Carboxyfluorescein-Sulforhodamine B

91. Electron transfer Intermolecular Electron transfer always occurs via collision and requires diffusion (O 2 will diffuse 7 nm in 10 ns in aqueous solution) maximum rate constant for bimolecular reaction is in the order of 4x10 10 Excited donor is a better donor, excited acceptor is a better acceptor

93. Markus theory for e-transfer : theory that describes how the rate constant of electron transfer depends on parameters such as orientation, ΔG, solvent reorganization, distance….

94. Kinetics of quenching The case of bimolecular quenching (stationairy) K is Stern-Volmer constant in l.mol -1 k d is the rate constant for deactivation without quenching Stern-Volmer equation

95. Kinetics of quenching The case of bimolecular quenching (time resolved) with k d is the rate constant for deactivation without quenching Stern-Volmer equation

96. Kinetics of quenching The case of intramolecular quenching Solving the equation leads to k d is the rate constant for deactivation without quenching Stern-Volmer equation or

97. Examples

101. Fluorescence polarization Anisotropy to study micro-viscosity in membranes and aggregation

102. Kinetics of quenching

103. Energy transfer Distance determination form the extend of transfer

104. Energy transfer R 0 = 5 nm

105. Photosynthesis Humans, animals, fungi, bacteria live by degrading molecules provided by other organisms…. Life on earth obviously could not continue indefinitely in this manner without an independent mechanism for synthesizing complex molecules from simple ones: the energy provided in this mechanism comes from the sun and is captured in the process of photosynthesis. Plants and other photosynthetic organisms fixe 10 11 tons/year of carbon in organic compounds (carbohydrate molecules, noted (CH 2 O)) from CO 2. But globally, the consumption is higher than the synthesis…. So, what will happen? CO 2 + H 2 O + light  (CH 2 O) + O 2  Important to understand the photosynthesis and how our activities affect it! Note: 1/3 of the fixed C is done by microorganisms in the oceans. Some bacteria also participate to the photosynthesis. Equilibrium constant: K= 10 -496  huge thermodynamic gradient!

106. http://www.life.uiuc.edu/govindjee/papers/milestones.html Porphyrin ring Chlorophyll structure c.f. TZ 7.5

107. The First Step: absorption of light In addition to chlorophyll, plants contain several pigments that absorb light The accessory pigments have antioxidant functions as well

108. EXCITATION light & heat light 3 POSSIBLE DECAY PATHWAYS e-e- excited pigment molecule 1. fluorescence 2. resonance energy transfer 3. successive resonance energy transfers neighboring pigment molecule e - donor e - acceptor +-+- After Alberts Fig. 14-47 Energy transfer after light absorption

109. Chlorophyll Pigment molecules Resonance transfer of light energy Electron acceptor “Special pair” of chl a molecules Carotenoid or other pigment Raven Fig 7-13; c.f. TZ 7.7

110. Note: for bacteria, the antenna systems are called LH-I and LH-II. They have this characteristic hollow cylinder shape. LH-I has a reaction center (RC) within this cylinder. LH- II has 9 bacteriochlorophylls outside the cylinder (to take the light) and 18 within the cylinder (to transfer the energy). 32 bacteriochlorophylls 18 + 9 bacteriochlorophylls

111. Events at the PS II reaction center c.f. TZ 7.24

112. Photosynthesis and aerobic respiration complete a cycle

113. Energy hopping Energy transfer in multichromoporic systems key-process in photosynthesis. The energy transfer process influenced by : - extend of coupling between the chromophores. - disorder (slow and fast fluctuations of the surrounding proteins )... Why investigate multichromophoric systems?

114. Energy hopping

117. Fluorescence decay analysis

118. Energy hopping

119. k hopp

120. Energy hopping k hopp = 4.6ns -1

121. Energy transfer

124. Fluorescence decay analysis

125. Cameleon protein YC3.1 Fluorescent indicators for measuring Ca 2+ concentration. - Energy donor : ECFP ECFP EYFP CaMM13 440 nm 475 nm ECFP EYFP 440 nm 530 nm - Energy acceptor : EYFP - Linker : calmodulin (CaM) + calmodulin-binding peptide M13 (myosin light chain kinase) Binding of Ca 2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer between the flanking GFPs. +4Ca 2+ -4Ca 2+

126. Definitions FRET: the excited donor transfers its energy to the acceptor via a dipole-dipole interaction. Requirements : - emission spectrum of donor and acceptor must overlap. - transition dipole moments of donor and acceptor must be sufficiently aligned. - distance between donor and acceptor must be such that probability of transfer is high. FRET can be detected by : - a decrease in donor decay time - a decrease in donor fluorescence intensity - an increase in acceptor fluorescence intensity - a change in fluorescence polarization - growing in component in acceptor decay

127. Absorption and emission spectra of EYFP  f (400 nm excitaiton) = 0.02 514 nm : deprotonated form.  f (500 nm excitaiton) = 0.61 400 nm : protonated form. - Absorption spectrum - Emission spectrum 528 nm : deprotonated form.  ESPT = 0.03

128. Excited-state photophysics of EYFP 560 nm 1 3.4 440 nm 0.9 0.006 0.1 0.06 a1a1  1 (ns) a2a2  2 (ns) 560 nm 1 3.4 excitation detection 400 nm 488 nm

129. Excited-state photophysics of EYFP 6 ps ESPT 60 ps,  = 0.03 3.4 ns A1*A1*A2*A2* I* B* A1A1 A2A2 IB ~ 480 nm~ 400 nm ~ 514 nm ~ 528 nm~ 480 nm - The A 2 * form having a conformation that allows ESPT, will relax to the I* state within 60 ps. - The A 1 * form will decay radiatively to its corresponding ground state, its fluorescence being quenched down to 6 ps by a non-radiative process.

130. Photophysics of ECFP 0.01 0.24 0.10 1.0 0.89 3.2 a2a2  2 (ns) a3a3  3 (ns) a1a1  1 (ns)

131. ECFP and EYFP as an energy transfer pair - The strong overlap of the emission spectrum of ECFP with the absorption spectrum of EYFP. Although displaying complicated photophysics, ECFP and EYFP still can be used to construct an energy transfer pair. - The relative high quantum yield of fluorescence of ECFP (  f = 0.4). - The mono-exponential decaying of fluorescence of EYFP when excited at the deprotonated band.

132. Emission spectra of YC3.1 ECFP EYFP ECFP EYFP +4Ca 2+ -4Ca 2+ I D : the integrated fluorescence intensity of the donor I A : the integrated fluorescence intensity of the acceptor  D : the fluorescence quantum yield of the donor  A : the fluorescence quantum yield of the acceptor  DA : the fluorescence quantum yield of the donor in the presence of acceptor Ca 2+ -binding YC3.1E = 0.29 Ca 2+ -free YC3.1E = 0.16

133. The distance between ECFP and EYFP R 0 : the critical transfer distance R : the distance between the donor and the acceptor k ET : the rate constant of energy transfer k f : the rate constant of donor in the absence of acceptor  DA : the fluorescence quantum yield of the donor in the presence of acceptor  2 : orientation factor n : the refractive index of the solvent N A : Avogadro’s number f( ) : the fluorescence spectrum of the donor normalized on the wavenumber scale  ( ) : the molar extinction coefficient of the acceptor at that wavenomber Ca 2+ -binding YC3.1R = 57 Å Ca 2+ -free YC3.1R = 65 Å

134. The distance between ECFP and EYFP ECFP EYFP ECFP EYFP 47 × 32 × 30 Å 24 Å 42 Å Ca 2+ -binding YC3.1R = 57 ÅCa 2+ -free YC3.1R = 65 Å >120 Å The estimated R value is consistent with the proposed structure. - Even for assuming the perfectly oriented transition dipole moment (  2 = 4), the efficiency of the energy transfer is estimated to be E = 0.027 if the protein adopt the most extended conformation (R = 120 Å). Relatively compact conformation of the protein construct, even in the Ca 2+ -free condition.