Fluorescence 2009 update

Transcription

1 XV 74 Fluorescence 2009 update Jablonski diagram Where does the energy go? Can be viewed like multistep kinetic pathway 1) Excite system through A Absorbance S 0 S n Excite from ground excited singlet ΔS = 0 could be any of them (FC overlap P ij ) must change dipole (very fast process, fsec) 2) If S n > S 1 often rapid decay/relax down (use VR, NR) S n S 1 called: IC internal conversion (ΔE ~ 0 fastest steps, goes to excited vib. level) 3) Options for release of energy in υ = 0 next step is a) NR - non-radiative decay to S 0 less probable implies IC to another singlet, S 0, but big separation Process: VR - Vibrational relaxation through collision vibrational energy taken to solvent (surround) relax to lowest vibrational state υ = 0 (relatively fast process < ns)

2 XV 75 b) Fluorescence emit photon to S 0 most probable for S 1 trap Fluorescence lifetime / quantum yield reflects probability of this process ns μs typical c) ISC cross over to triplet -- intersystem crossing In triplet VR to υ = 0 again trap energy Phosphorescence much slower (ΔS 0) aided by heavy atoms (spin-orbit) Fluorescence intensity depends on how molecules are excited and on the probablity of transition to ground state. It is a kinetic process compete between pathways in Jablonski diagram, typically excite by absorbance Competition between fluorescence, non-radiative decay, and intersystem crossing. First order process: -d[m*]/dt = k d [M*] Lifetime: τ = 1/k d if only fluorescence: τ 0 = 1/k f Other processes take away excitation, --lead to shorter observed lifetimes -d[m*]/dt = k f [M*]+ k nr [M*]+ k Q [M*][Q] = k d [M*] where k nr [non-radiative decay], k Q [quenching] observed lifetime: τ = 1/(k f + k nr + k Q [Q]) = 1/k d Quantum yield ratio: photons fluoresce / photon absorb φ f = # photon (F)/# photon (A) = k f [M*]/ k d [M*] = τ/τ 0

3 XV 76 Quantum Mechanics role 1) Quantum mechanics used to describe excited states much less accurate than for vibrations (excited st.) requires a surface not just single geometry calculations need configuration interaction states become mix of configurations: (σ) n (π) m idea need to change orbitals of electron states mix different orbital configuration impact calculations large and less accurate 2) Quantum mechanics used to describe which vibronic excitation are allowed ΔS = 0 Electric field cannot change spin (Phosphoresce mix spins with spin-orbit coupling) Dipole must change: A ~ ψ ex * μ el ψ g dτ 2 integral zero if ψ ex and ψ g same dipole Δυ = 0, ±1, ±2 no restriction on υ - sym. modes A: Absorb: most transition start υ g = 0 (most population) F: Fluorescence is same but υ ex = 0 by relaxation (VR) 3) Transitions seen usually determined by symmetry group theory tool for organizing symmetry useful in small molecules (Chem 444) Biomolecules less use no symmetry use correlation to small molecule components

4 Absorption and Fluorescence sample the same states, but processes give fluorescence added dimension Intensity dipole strength D ij = μ ij 2 = 0.92x10-38 (ε/ν)dν (esu-cm) 2 [or x10-2, and units of Debye 2,1D=10-18 esu-cm] XV 77 Absorption detect same photons as excite, A = -log I/I 0 To go beyond average result, need to orient molec. use polarization (next sect.), lifetime no meaning Sensitivity limited difference of big #s: [log I log I 0 ] Fluorescence excite different photon than detect Transfer of energy between states kinetic process Polarization can detect change orientation Can go outside of chromophore - intramolecular FRET fluorescence resonant energy transfer (Engel 19.13) Efficiency of transfer from Donor (D) and Acceptor (A) E ff = k T /(k T + k f ) compare rates: F (k f ) & transfer(k T ) depends on distance and spectral overlap Forster transfer rate: k FRET = 1/τ D (R 0 /r) 6 Where: τ D = Donor lifetime, r = distance D A R 0 = experimental param., rate transfer = rate decay Result: FRET can provide a spectroscopic ruler For molecules in nm range note 6 th power! Bio-applications see Engel Ch tag parts of protein or DNA with fluorphores (D and A) observe relative intensity of fluorescence from A or better change in its lifetime, τ = 1/k FRET

5 Calibrate with known lengths eg dsdna, poly-pro Can use dyes, or proteins Tryptophan to dye XV 78 Quenching process deactivates excited state/reduce F Collision can take away energy or cause molecule to change states to non-fluorescent one e.g. O 2 : M*(sing) + O 2 (trip) M*(trip) + O 2 (sing) Process, multistage kinetic path M + hν M* (excitation) M* M + hν (fluorescence) M* + Q M + Q (quenching) Rates: no quenching: φ 0 f = k f /(k f +k nr +k ISC ) With quenching: φ f = k f /(k f +k nr +k ISC +k Q [Q]) Stern-Volmer relation: φ 0 f / φ f = 1 + k Q [Q]/ (k f +k nr +k ISC ) = 1 + K[Q] lifetime as well: φ 0 f / φ f - 1 = k Q τ [Q] with τ = 1/(k f +k nr +k ISC ) plot: F 0 /F vs. [Q] and slope is k Q τ Can use this to sense exposure of fluorphores to surface Reflects change in environment e.g. unfold tertiary Polarization Since μ is vector fixed on molecule, E-field can interact with molecule differently if change E-orientation Transitions can be allowed for x,y,z orient μ in molecule e.g. s p x in H-atom, allowed by E x excite μ x, π π* in ethylene, C 2 H 4, polarize along C=C bond

6 XV 79 gas or solution no impact / average out solid can orient molecule crystal used for small molecule Alternative dissolve in oriented material a) liquid xtal Net orientation long axis of inserted molecule favor orientation b) lipid membrane composed of charged head groups and alkyl tails, bilayer form: -- self-assembles in layers alkyl interior favor hydrophobic e.g. Helices orientation, surface, alkyl tails eg trans membrane protein / peptide hydrophilic surface can bind charges, e.g. amphipathic helices lay on surface

7 XV 80 c) Flow long molecules orient to flow Works well for DNA, fibers, etc. d) surfaces, and reflection, provide alternate - sense polarization, s&p Useful if chromophore absorbing species has different absorbance with one polarization called dichroism (linear) can use for analysis of orientation in fluorescence if excite with one polarization can observe emission in and orientation fluorescence anisotropy degree of motion / flexibility ideal - measure change in polarization with time r = (I - I )/(I + 2I )

8 XV 81 Fluorescence anisotropy Changes in fluorescence anisotropy 0.08 A ph6.8 DPH TMA-DPH DMPG B ph4.6 DOPG DSPG DMPG DOPG DSPG

9 XV 82 High density lipo-protein can make a discoid with lipid Polarized IR can tell orientation of the helices In plane Out of plane

10 XV 83 Circular Polarization if 2 waves displaced by λ/4 along z combine get rotation of E as propagate (helix in space) Circular Polarization Right or Left Now molecule sees both linear polarizations (x + y) but due to the rotation between them at ν has different selection rules Trick measure difference: ΔA = A L A R

11 XV 84 Circular Dichroism Theoretically this ~ R = Im [( ψ ex m ψ g ) ( ψ ex μ ψ g )] m electronic dipole operator μ magnetic dipole operator μ m 0 only for chiral molecules eg chiral / asymmetric C / no plane or center of symmetry Perfect for biology all bio-molecular, chiral i.e. proteins L AA DNA chiral ribose sugars several centers lipids well

12 Measurement of CD is most widely used for protein secondary structure most intense α-helix 222 & 207nm weaker β-sheet, neg 215, pos 200 XV 85

13 XV 86 DNA typical band patterns vary Big success: B Z differ: right left handed helices Sugars problem, absorbance bands in VUV Lipids -- similar issues ORD - like measuring index of refraction no absorption Can measure optical rotation in clear samples φ = (π/λ)(n L n R ) where n L & n R index in circ. Light circular birefringence CD is absorption spectra, so need absorption band ΔA = A L A R but can be measured as ellipticity θ(degrees) = ΔA alternate: - molar ellipticity scale ( l path(cm or dm)): [θ] = 100 θ/cl = 3300 Δε Can convert CD ORD Transform: integrate over all λ

14 XV 87 In IR can do VCD, called Vibrational Circular Dichroism Signals smaller (need more concentration) but differentiation between states/conformations is higher VCD measures same transitions as IR, but has shape/sign Patterns for VCD discriminate helices, sheets and coils Also distinguish helices, turns and other structures Coil shown to be characterized by left hand turns due to similarity with poly L-Pro II helices

15 XV 88 Derivative shape from coupling of dipoles (CD and VCD) DNA VCD - base region sensitive to G-C ratio, not PO 2 Easily sense B- and Z-form DNA, A- similar to B- form Triplex DNA has unique pattern

16 Also can use isotopes to localize structural information XV 89