Biointerfacial Characterization

Fluorescence Spectroscopy
 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.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
 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 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)  E / kT
Re
• 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
Fluorescence Probing: Solvation; Reorientation
polarization
anisotropy
I || (t )  I  (t )
r (t ) 
I || (t )  2 I  (t )
Time-dependent
fluorescence
Stokes shift

hlaser

 (t )  ()
C (t ) 
 (0)  ()
Solvation Coordinate
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.
Figure 1.13
•  is the fluorophore emission rate and
the nonradiative decay to So rate is knr.
• The fluorescence quantum yield is

given by Q    k nr

• Excited state lifetime: typically 10 ns,

1

  knr
7
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.
I I
• Fluorescence anisotropy r is defined by:

r  P

I  2I
P 
I I
• Polarization is defined by P: P  I  I P 

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
0.10
0.05
60 0.00
-0.05
-0.10
50 0.4

40 III 0.3

r(t)
3
x10

30 I 0.2
20
0.1
10

0 0.0

0 10 20 30 40 5 10 15 20 25
ns
ns

•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/0)b  r
(above: Coumarin 343-/Na+ in 25% aqueous F88 triblock copolymer)
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
Aggregate Structures in
PEO-PPO-PEO Solutions

random coil micelles hydrogels

(unimer) (above cmc/cmT) (above cgc/cgT)

Increasing Temperature (concentration)

R.K. Prud’homme et al Langmuir 1996 (12) 4651 (cubic gel structure)

13
 Coumarin Fluorescence Probes

C153 C102 C343-/Na+

CF3 CH3 O

O-/Na+

N O O N O O N O O

Localizes in PPO Localizes in Located primarily

hydrophobic/dry PPO/PEO regions in wet phases
core (water?)

clogP = 4.08 clogP = 3.67 clogP = -1.09

14
 Fluor. excitation and emission spectra
O

O-/Na+

N O O

•Aq. PEO109-PPO41-PEO109
•5 w/v % solution forms
micelles
CH3
•Probes localize in different
regions N O O

–Experience different
electrical environments

CF3

N O O

15
 CF3
~17 nm

N O O

C153

7.6-10.4 nm
16
N. J. Jain et al. JPCB 1998 (102), 8452.
C102
CH3

N O O

17
C343-/Na+
O

O-/Na+

N O O

18
Temperature Dependent Emission Shifts

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
C343 — anion weakly sensitive to microphase transition
19
Fluorescence Probing:
Reorientation

Detection of
emission de-
polarization reports
on micro-viscosity

hlas
polarization
er
anisotropy
I|| (t )  I  (t )
r (t ) 
I|| (t )  2 I  (t )

20
Simultaneously fit Anisotropy, r(t), double exponential
reorientation 21
 5 w/v% F88
C153
 Local friction (qrot) increases by
3.5 times over the cmT
Extremely sensitive to
environmental changes in PPO
core
C102
qrot increases by ~ 2 times over cmT
Shifted to slightly higher T
25 w/v% F88
Distributed in multiple
environments
C343-/Na+
qrot decrease scales roughly with
decreasing macroscopic viscosity
Mainly in bulk water/hydrated
PEO regions
Grant, Steege, DeRitter, Castner
J. Phys. Chem. B, 2005, 109, 22273. 22
 q  58.1 0.96
Pol
rot

q  34.8  0.63
NPol
rot

C153 local friction Rheology estimates Tgel

increases from 14– 890 cp macroscopic viscosity ~107 cP
in gel forming
concentration (25 w/v%)
Calculated from Maroncelli et al J. Phys. Chem. A, 1997, (101) 1030 23
 Principles of Time-Correlated Single-Photon Counting

(TCSPC)

see text by Wolfgang Becker,

Chemistry Library
25