125:583 Biointerfacial Characterization Oct. 2 and 5, 2006 Fluorescence Spectroscopy

1. 125:583Biointerfacial CharacterizationOct. 2 and 5, 2006Fluorescence Spectroscopy Prof. Ed Castner Chemistry Chemical Biology Prof. Prabhas Moghe Chemical & Biochemical Engineering

2. Introduction to Fluorescence • Luminescence: emission of photons from electronically excited states of atoms, molecules, and ions. • Fluorescence: Average lifetime from <10—10 to 10—7 sec from singlet states. • Phosphorescence: Average lifetime from 10—5 to >10+3 sec from triplet excited states.

3. Reference Reading • B. Valeur, “Molecular Fluorescence: Principles and Applications”, Chem. Library, call number: QD96.F56V35 2002 • J. Lakowicz, “Principles of Fluorescence Spectroscopy”, Chem. Library,call number: QD96.F56L34 1999 • W. Becker, “Advanced Time-Correlated Single-Photon Counting Techniques, Chem. Library,call number: QC793.5.P422B43 2005

4. Why Use Fluorescence Spectroscopy? • Sensitivity to local electrical environment • polarity, hydrophobicity • Track (bio-)chemical reactions • Measure local friction (microviscosity) • Track solvation dynamics • Measure distances using molecular rulers: fluorescence resonance energy transfer (FRET)

5. Photophysics: Jablonski Diagram • Photoexcitation from the ground electronic state S0 creates excited states S1, (S2, …, Sn) • Kasha’s rule: Rapid relaxation from excited electronic and vibrational states precedes nearly all fluorescence emission. • (track these processes using femtosecond spectroscopy) • Internal Conversion: Molecules rapidly (10-14 to 10-11 s) relax to the lowest vibrational level of S1. • (This is why DNA doesn’t emit much fluorescence) • Intersystem crossing: Molecules in S1 state can also convert to first triplet state T1; emission from T1 is termed phosphorescence, shifting to longer wavelengths (lower energy) than fluorescence. Transition from S1 to T1 is called intersystem crossing. Heavy atoms such as Br, I, and metals promote ISC. 5

6. polarization anisotropy Time-dependent fluorescenceStokes shift hnlaser Solvation Coordinate Fluorescence Probing: Solvation; Reorientation 6

7. Fluorescence Lifetimes and Quantum Yields • Quantum yield: ratio of the number of emitted photons to the number of absorbed photons. • Fluorophores with highest quantum yields exhibit the brightest emission (e.g., rhodamines), when normalized to absorption strength. • G is the fluorophore emission rate and the nonradiative decay to So rate is knr. • The fluorescence quantum yield is given by • Excited state lifetime: typically 10 ns, Figure 1.13 7

8. Fluorescence Polarization Anisotropy • Information about the size and shape of proteins or rigidity of various molecular environments. • Fluorophores preferentially absorb photons whose electric vectors are aligned parallel with transition moment of the fluorophore. In an isotropic solution, fluorophores are oriented randomly. Upon excitation with polarized light, one selectively excites those fluorophore molecules whose absorption transition dipole is parallel to the electric vector of the excitation. This selective excitation results in partially oriented population of fluorophores and in partially polarized fluorescence emission. • Fluorescence anisotropy r is defined by: • Polarization is defined by P: • Where I|| and I are the fluorescence intensities of the vertically (||) and horizontally( ) polarized emission, when the sample is excited with vertically polarized light. 8

9.  Rotational Dynamics: Anisotropy r(t) • r(t) = distribution of relaxation times, relates to rotational diffusion • Fit equation with a multiple or a stretched exponential • Stretched Exponential Fit: r(t) = (r0-r)exp(-t/t0)b + r • (above: Coumarin 343-/Na+ in 25% aqueous F88 triblock copolymer) 9

10. Instrumentation: Time-Integrated Spectrofluorometer 10

11. Intrinsic Fluorophores • tetrapyrroles: • hemes • chlorophylls • pheophytins • carotenoids 11

12. Extrinsic fluorophores • rhodamines • fluoresceins • coumarins • carbocyanine dyes • aromatic hydrocarbons and derivatives: • pyrenes, perylenes, anthracenes • See Invitrogen Molecular Probes catalog 12

13. Aggregate Structures in PEO-PPO-PEO Solutions micelles(above cmc/cmT) hydrogels (above cgc/cgT) random coil (unimer) Increasing Temperature (concentration) 13 R.K. Prud’homme et al Langmuir1996 (12)4651 (cubic gel structure)

14. Coumarin Fluorescence Probes C153 C343-/Na+ C102 Localizes in PPO/PEO regions (water?) Located primarily in wet phases Localizes in PPO hydrophobic/dry core clogP = 3.67 clogP = -1.09 clogP = 4.08 14

15. Fluor. excitation and emission spectra Aq. PEO109-PPO41-PEO109 5 w/v % solution forms micelles Probes localize in different regions Experience different electrical environments 15

16. ~17 nm C153 7.6-10.4 nm N. J. Jain et al. JPCB1998(102), 8452. 16

17. C102 17

18. C343-/Na+ 18

19. 5 w/v% 25 w/v% Temperature Dependent Emission Shifts • C153 and C102 — Blue Shift –Polar  Non-polar • C102 — Blue shift at ~2-4 °C higher than C153 • Distributed between PPO and the PEO-PPO interface • C343—anion weakly sensitive to microphase transition 19

20. polarization anisotropy hnlaser Fluorescence Probing: Reorientation Detection of emission de-polarization reports on micro-viscosity 20

21. Simultaneously fit Anisotropy, r(t), double exponential reorientation 21

22. 5 w/v% F88 25 w/v% F88 • C153 • Local friction(qrot)increases by3.5 times over the cmT • Extremely sensitive to environmental changes in PPO core • C102 • qrotincreasesby~ 2 times over cmT • Shifted to slightly higher T • Distributed in multiple environments • C343-/Na+ • qrotdecrease scales roughly with decreasing macroscopic viscosity • Mainly in bulk water/hydrated PEO regions Grant, Steege, DeRitter, CastnerJ. Phys. Chem. B,2005, 109, 22273. 22

23. C153 local friction increases from 14– 890 cp in gel forming concentration (25 w/v%) Rheology estimates Tgelmacroscopic viscosity ~107 cP 23 Calculated from Maroncelli et alJ. Phys. Chem. A, 1997,(101) 1030

24. Principles of Time-Correlated Single-Photon Counting (TCSPC) see text by Wolfgang Becker,Chemistry Library

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