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

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.

Reference Reading B. Valeur, “Molecular Fluorescence: Principles and Applications”, Chem. Library, call number: QD96.F56V J. Lakowicz, “Principles of Fluorescence Spectroscopy”, Chem. Library, call number: QD96.F56L W. Becker, “Advanced Time-Correlated Single- Photon Counting Techniques, Chem. Library, call number: QC793.5.P422B

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)

Photophysics: Jablonski Diagram Photoexcitation from the ground electronic state S 0 creates excited states S 1, (S 2, …, S n ) 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 ( to s) relax to the lowest vibrational level of S 1. –(This is why DNA doesn’t emit much fluorescence) Intersystem crossing: Molecules in S 1 state can also convert to first triplet state T 1 ; emission from T 1 is termed phosphorescence, shifting to longer wavelengths (lower energy) than fluorescence. Transition from S 1 to T 1 is called intersystem crossing. Heavy atoms such as Br, I, and metals promote ISC. 5

Fluorescence Probing: Solvation; Reorientation Solvation Coordinate h laser Time-dependent fluorescence Stokes shift polarization anisotropy 6

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.  is the fluorophore emission rate and the nonradiative decay to S o rate is k nr. The fluorescence quantum yield is given by Excited state lifetime: typically 10 ns, Figure

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

Rotational Dynamics: Anisotropy r(t) = distribution of relaxation times, relates to rotational diffusion Fit equation with a multiple or a stretched exponential Stretched Exponential Fit: r(t) = (r 0 -r  )exp(-t/  0 )   r  (above: Coumarin 343-/Na+ in 25% aqueous F88 triblock copolymer) r(t)  9

Instrumentation: Time-Integrated Spectrofluorometer 10

Intrinsic Fluorophores tetrapyrroles: hemes chlorophylls pheophytins carotenoids 11

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

random coil (unimer) micelles (above cmc/cmT ) hydrogels (above cgc/cgT ) Increasing Temperature ( concentration) R.K. Prud’homme et al Langmuir 1996 (12) 4651 (cubic gel structure) Aggregate Structures in PEO-PPO-PEO Solutions 13

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

Fluor. excitation and emission spectra Aq. PEO 109 -PPO 41 -PEO w/v % solution forms micelles Probes localize in different regions –Experience different electrical environments 15

C153 ~17 nm nm N. J. Jain et al. JPCB 1998 (102),

C102 17

C343 - /Na + 18

 C343 — anion weakly sensitive to microphase transition 5 w/v% 25 w/v%  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 Temperature Dependent Emission Shifts 19

Fluorescence Probing: Reorientation polarization anisotropy h las er Detection of emission de- polarization reports on micro-viscosity 20

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

 C153  Local friction (  rot ) increases by 3.5 times over the cmT  Extremely sensitive to environmental changes in PPO core  C102   rot increases by ~ 2 times over cmT  Shifted to slightly higher T  Distributed in multiple environments  C343-/Na+   rot decrease scales roughly with decreasing macroscopic viscosity  Mainly in bulk water/hydrated PEO regions 5 w/v% F88 25 w/v% F88 Grant, Steege, DeRitter, Castner J. Phys. Chem. B, 2005, 109,

C153 local friction increases from 14– 890 cp in gel forming concentration (25 w/v%) Rheology estimates T gel macroscopic viscosity ~10 7 cP Calculated from Maroncelli et al J. Phys. Chem. A, 1997, (101)

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

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