Presentation to accompany my report for my oral examination. Details background of fluorescence upconversion techniques, development of measurement systems for release of a metal cation and minimization of diffusion distribution in solutions.

1. METAL ION BURST:
EXAMINING METAL ION
DIFFUSION USING
ULTRAFAST FLUORESCENCE
SPECTROSCOPY IN REVERSE
MICELLES
CHELSEY CROSSE
ORALS PRESENTATION
APRIL 29, 2014

2. to develop methods
to measure the
impact of metal ions
on a wide range of
timescales.
1
RESEARCH FOCUS:
Example: Proposed work with Dr. Crans
observing interaction of Cu2+ and β-amyloid. All
current work is directed in this capacity.
Illustration courtesy of Alzheimer’s Disease Research, a program of the American Health Assistant Foundation

3. A STEP BACK:
TIME RESOLVED SPECTROSCOPY
2
Initialization
t0
t
Changes Equilibrium
…
0
1
2
3
0 5 10 15 20
Absorbance
(~620nm)
Time
Example Kinetic Trace at Single
Wavelength
0
0.2
0.4
0.6
0.8
1
400 500 600 700
Absorbance
(t~8)
Wavelength (nm)
Example Spectrum at Single Time

4. • Rate of observation
• Rate of initialization
3
LIMITS OF TIME RESOLVED
SPECTROSCOPY
t0
1s
Pouring
Human Eye
Photolysis Release
Optical Gating
Highest Possible Time Resolution
1ps
(approximate temporal orders)
Micro Channel Plate (electronic limit)
…

5. pH jump
• Photoacids excited by
femtosecond pulses
• Releases H+
• Regenerates
• Well characterized release
dynamics
Caged-metal organic
complexes
• Cages photolysed by
femtosecond pulses
• Releases metal ion
• Does not regenerate
• Release dynamics not
characterized on fs scale to
our knowledge
4
PHOTOLYSIS RELEASE
TECHNIQUES
HA hn
¾ ®¾ A-
+H+ time
¾ ®¾ HA
AMB hn
¾ ®¾ A+M +B
M2+ M2+
hn
H+
hn
H+
Donten, M. L., Hamm, P., & VandeVondele, J. (2011). A Consistent Picture of the Proton Release Mechanism of oNBA in
Water by Ultrafast Spectroscopy and Ab Initio Molecular Dynamics. The Journal of Physical Chemistry B , 115, 1075-1083.
First Step

6. 5
METAL ION DETECTION BY
FLUORESCENT PROBE
1. Photolysis
2. Diffusion
3. Interaction
1. Photolysis
2. Diffusion
3. Interaction
1. Photolysis
2. Diffusion
3. Interaction
1. Photolysis
2. Diffusion
3. Interaction
(1) hnM2+
(3) Interaction
M2+
Probe
(2) DiffusionM2+
M2+

7. 6
Requirements:
• Ultrafast probe technique
• Well characterized probe
• Limited diffusion distance
(1) hnM2+
M2+
Probe
M2+
M2+
METAL ION DETECTION BY
FLUORESCENT PROBE

8. • Energy of electronic
states depends on:
• Molecule geometry
• Interactions with
environment
• Changes can be
observed as:
• Changes in fluorescence
intensity
• Shifts in fluorescence
spectra
7
TIME RESOLVED FLUORESCENCE
ON MOLECULAR TIMESCALE
System Coordinate
t
t
FluorescenceIntensity

9. Detection of fluorescence
signal using non-linear
optical gating.
8
FLUORESCENCE
UPCONVERSION
wgated =wfl +wgate
Non-Linear Crystal
Gated
Fluorescence Signal (ωgated)

10. Portion of fluorescence
signal sampled can be
changed by:
• Changing gate arrival
time  temporal region
• Changing crystal angle
 spectral region
9
FLUORESCENCE UPCONVERSION:
TEMPORAL RESOLUTION
Non-Linear Crystal
Gated
Fluorescence SignalIntensity
Crystal Out
Crystal In

11. • Each frequency requires
realignment of crystal
angle
• Kinetic traces can be
used to reconstruct
temporal fluorescence
spectra
10
FLUORESCENCE UPCONVERSION:
EXAMPLE DATA
Zhang, X. -X.; Würth, C.; Zhao, L.; Resch-Genger, U.; Ernsting, N. P.; Sajadi, M. Femtosecond broadband fluorescence
upconversion spectroscopy: Improved setup and protometric correction. Review of Scientific Instruments 2011, 82,
063108.
t
FluorescenceIntensity

12. 11
FLUORESCENCE UPCONVERSION:
TEMPORAL RESOLUTION
BBO
Sample
Nd:Vanadate
Pump Laser
Mode-locked
Ti:Sapphire Laser
Optical
Delay Stage
BBO
Elliptical
Mirror
Monochromator
PMT
LabVIEW VI
Used successfully by
Levinger group to
examine water
environments
I have rebuilt most of this
system, repairs &
replacements have
prevented completion
Gate
Excitation
Gated
Signal

13. I have experience aligning
necessary optical systems:
• Assembled &
successfully mode-locked
Ti:Sapphire oscillator
system
• Used & adjusted FROG
system
• Designed & built other
non-linear system
12
FLUORESCENCE UPCONVERSION:
PERSONAL EXPERIENCE

14. 13
RELATIVE
TIMESCALES
• Fluorescence lifetime: ~10-9 s
• Diffusion of metal ion: 10-13-10-6s
(1) hnM2+
M2+
Probe
M2+
M2+

15. 14
Requirements:
 Ultrafast probe technique
• Well characterized probe
• Limited diffusion distance
(1) hnM2+
(3) Interaction
M2+
Probe
(2) DiffusionM2+
M2+
METAL ION DETECTION BY
FLUORESCENT PROBE

16. Ideal fluorophore:
1. Inert
2. Selective & sensitive
3. Detectable by our system, currently:
• Absorption: 400-450nm
• Emission: 450-500nm
• Detectable intensity
15
FLUOROPHORE
SELECTION

17. Initial characterization:
• Steady State
Fluorescence
• Fluorescence Lifetime
(TCSPC)
Have been able to observe
quenching by Cu2+.
16
COUMARIN 343 (C343)
Previously used successfully by Levinger group in
fluorescence upconversion studies.

18. 0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
450 460 470 480 490 500 510 520 530 540 550
FluorescenceIntensity[counts]
Wavelength (nm)
Steady State Fluorescence Spectra for Solutions of C343 excited by 372nm NanoLED with
Varying Concentrations of CuSO4
0.0E+00
5.5E-06
1.1E-05
2.2E-05
4.4E-05
1.1E-04
2.2E-04
Concentration CuSO4 (M)
17
C343: STEADY STATE
FLUORESCENCE
0
1
2
3
4
5
0.0E+00 1.0E-04 2.0E-04
Thousands
Concentration CuSO4 [M]
Peak Fluorescence
Intensity

19. 18
C343: FLUORESCENCE
LIFETIME
Fluorescence Lifetime Measurements of 2.2E-5 [M] C343
with Varying Concentration of CuSO4

20. Used to determine complex
stoichiometry.
1. Measure absorbance of
mixture solution at
different mole fractions
2. Subtract the absorbance
of the pure compounds
in solution at the
concentration measured
19
JOB PLOT
Harris Quantitative Chemical Analysis Fig. 19-8
A(PX, x)= A(solution, x)- A(P,[P])- A(X,[X])

21. C343: BEER’S LAW
20
In cooperation with Angela Warner.
A =ebc
y = 0.253x
R² = 0.9636
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.5 1 1.5 2 2.5
Absorbance
Concentration of C343 (10-5M)
Beer's Law Plot C343 at 409 nm
y = 0.3417x
R² = 0.9997
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.5 1 1.5 2 2.5
PeakAbsorbance
Concentration (10-5M)
Beer's Law Plot of Tryptophan at 212 nm
C343:
R² = 0.96364

22. JOB PLOT:
TRYPTOPHAN
21
0
0.005
0.01
0.015
0.02
0.025
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
CorrectedAbsorbance
Mole Fraction of Cu2+
Job Plot of Tryptophan with CuSO4
Corrected Abs
@204nm
Corrected Abs
@212nm
Corrected Abs
@219nm
Corrected Abs
@230nm
Corrected Abs
@246nm
Corrected Abs
@279nm
Corrected Abs
@300nm
In cooperation with Angela Warner.
0
0.2
0.4
0.6
0.8
200 250 300 350
Absorbance
Wavelength (nm)
Absorbance Spectrum
of Tryptophan
(2.2 × 10-5 M)

23. 22
Requirements:
 Ultrafast probe technique
 Well characterized probe
• Limited diffusion distance
(1) hnM2+
(3) Interaction
M2+
Probe
(2) DiffusionM2+
M2+
METAL ION DETECTION BY
FLUORESCENT PROBE

24. • Open solution:
• Sensor-ion interaction
determined by Brownian
diffusion
• Large probability distribution
• Limited measurement
resolution
• Confined environment:
• Sensor-ion interaction
probability enhanced by
proximity
• Increased reaction times have
been shown previously
• Choose reverse micelles
(RMs)
Levinger group has
expertise in characterization
& preparation.
23
CHARACTERIZATION OF
METAL ION RELEASE
M2+
Probe
M2+
Probe

25. 24
 Ultrafast probe technique
• Fluorescence upconversion
• Currently being reassembled
 Well characterized probe
• Analytical characterization techniques have been validated
• Still in the process of testing candidates
 Limited diffusion distance
• Confinement in reverse micelles
• Relying on expertise of Levinger group
METAL ION DETECTION BY
FLUORESCENT PROBE

26. Immediate:
• Identify fluorophore
• Build upconversion
experiment
• Characterize full
fluorophore system using
fluorescence
upconversion
Future:
• Extend upconversion to
metal ion burst
• Measure metal ion
dynamics in interesting
systems
25
GOALS
M
Probe

27. Dr. Nancy Levinger
Dr. Debbie Crans
Angela Warner
Ben Wiebenga-Sanford
All the Levinger & Crans
group folks
Barisas (use of the IBH)
CIF
Jenee Cyran & Laura Tvedke
26
ACKNOWLEDGEMENTS

28. 27
ANATOMY OF A
REVERSE MICELLE
• Organic solvent
• Aqueous suspension
• Surfactant molecules

29. 28
REVERSE MICELLE: TERNARY
PHASE DIAGRAM
• Size
• Described in number of water
molecules (w0) in each RM
• Easily adjusted by changing
proportions of solution
• Well documented
characteristics
• Rate enhancements
observed in confined
reaction systems
Magalhaes – Phase diagram of a lyotropic mixtrue sodium bis (2 ethylexyl) sulfosuccinate-codecanol-water:…(1998)
Confinement papers from Levinger (78-80 PRF)

30. • Photolytic release of
metal ions into solution
• Complex dissociation has
been observed to be
faster than 10 s
• Variety of compounds
with different optical
absorbances
• Diffusion time of metal
ion has yet to be
characterized
29
CAGED METAL
ORGANIC COMPLEXES
H. M. D. Bandara, D. P. Kennedy, E. Akin, C. D. Incarvito, and S. C. Burdette, "Photoinduced Release of Zn2+ with ZinCleav-1: a Nitrobenzyl-Based Caged Complex",
Inorganic Chemistry 48 (17), 8445-8455 (2009).
H. M. D. Bandara, T. P. Walsh, and S. C. Burdette, "A Second-Generation Photocage for Zn2+ Inspired by TPEN: Characterization and Insight into the Uncaging Quantum Yields
of ZinCleav Chelators", Chemistry-a European Journal 17 (14), 3932-3941 (2011).
K. L. Ciesienski and K. J. Franz, "Keys for Unlocking Photolabile Metal-Containing Cages", Angewandte Chemie-International Edition 50 (4), 814-824 (2011).
K. L. Ciesienski, K. L. Haas, and K. J. Franz, "Development of next-generation photolabile copper cages with improved copper binding properties", Dalton Transactions 39 (40), 9538-9546 (2010).

31. Because we are observing
quenching effects, we will be
working in lower intensity
regions.
It may be necessary to
increase instrument
sensitivity.
OPAGE would gate signal in
an OPA crystal to account
for this
It has been tried before, but
have been some challenges
30
OPAGE

32. 31
C343 CHARACTERIZATION:
QUALITATIVE FLUORESCENCE
0
200000
400000
600000
800000
1000000
1200000
1400000
440 460 480 500 520 540
Intensity
Emission [nm]
0 drops
1 drop 0.01 M
2 drops 0.01 M
1 drop 0.1M
2 drops 0.1M
3 drops 0.1M
1 drops 1M
2 drops 1M
3 drops 1M
Fluorescence Measurements of 2.2E-5 M C343 with Varying
Concentrations of CuSO4 Excited at 400 [nm]
Amount of CuSO4
added

33. JOB PLOT:
TRYPTOPHAN
32
0
0.005
0.01
0.015
0.02
0.025
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Absorbance
Mole Fraction of Cu2+
Job Plot of Tryptophan with CuSO4
Corrected Abs
@204nm
Corrected Abs
@212nm
Corrected Abs
@219nm
Corrected Abs
@230nm
Corrected Abs
@246nm
Corrected Abs
@279nm
Corrected Abs
@300nm
EVERYONE LOOK HOW AWESOME ANGELA IS!!!

34. 33
C343
CHARACTERIZATION:
ABSORPTION
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
300 350 400 450 500 550
Absorbance
Wavelength [nm]
Absorbance of 2.2E-5 [M] C343 Solutions with Varying
CuSO4 Concentrations
0.0E+00
1.7E-04
3.4E-04
5.0E-04
6.7E-04
8.4E-04
1.0E-03
1.2E-03
Concentration*
of CuSO4 [M]
*Assumed molarity of a drop of CuSO4 solution in 2 [mL] of C343 = 1.7E-4
Isobestic Point?

35. FWHM: ~25 nm
34
EXAMPLE NANOLED
SPECTRUM
0
1000
2000
3000
4000
5000
6000
7000
250 300 350 400 450 500 550 600 650 700 750 800
Counts
Wavelength (nm)
372nm NanoLED Spectrum

36. FLUORESCENCE
LIFETIME FITS OF C343
AND CU2+
35
C343
2.5E-5M
0.01M CuSO4
3 drops
3.35E-3M
0.1M CuSO4
1 drop
1.12E-2M
0.1M CuSO4
2 drops
2.24E-2M
1M CuSO4
1 drop
0.112M
Single
exp.
Double
exp.
Single
exp.
Double
exp.
Single
exp.
Double
exp.
Single
exp.
Double
exp.
1 [s] 4.14E-9 4.19E-9 2.18E-9 4.00E-9 2.12E-10 3.84E-9 2.45E-10 3.55E-9 2.16E-10
2 [s] --- --- 4.24E-9 --- 4.03E-9 --- 3.89E-9 --- 3.64E-9
A -0.502 -0.199 -0.282 -0.152 -0.209 0.133 4.82E-2 0.202 0.103
B1 0.118 0.119 5.77E-3 0.117 6.75E-3 0.117 3.97E-2 0.115 0.139
B2 --- --- 0.115 --- 0.114 --- 0.112 --- 0.107
 2 1.385 1.380 1.358 1.398 1.120 1.687 1.210 2.935 0.984

37. Point at which two chemical
species have the same molar
absorptivity, or are linearly
related.
Can be used to tune
absorbance for total reaction
because it remains constant
over the entire reaction.
36
ISOSBESTIC POINT
Wikipedia. http://en.wikipedia.org/wiki/File:Bromocresol_green_spectrum.png Created 03/02/2006, Accessed 04/17/14

38. 37
PH JUMP EXAMPLE
Donten, M. L., Hamm, P., & VandeVondele, J. (2011). A Consistent Picture of the Proton Release Mechanism of oNBA in
Water by Ultrafast Spectroscopy and Ab Initio Molecular Dynamics. The Journal of Physical Chemistry B , 115, 1075-1083.

39. 38
TIMESCALES OF
CHEMICAL PROCESSES

40. Optical component
measured delay times:
• SHG crystal: 0.36 mm
(108 fs)
• Microscope slide: 0.7 mm
(208.8 fs)
• Sapphire window: 2.39
mm (717 fs)
• Lens set 1: 21 mm
(6301.2 fs)
• Lens set 2: 3.74 mm
(1122 fs)
39
AUTOCORRELATOR & OPTICAL
DELAY MEASUREMENTS

41. 40
R928 SPECTRAL
RESPONSE

42. 41