Post on 18-Apr-2020
TIME-RESOLVED OPTICAL SPECTROSCOPY
Petar LambrevLaboratory of Photosynthetic MembranesInstitute of Plant Biology
The Essence of Spectroscopy
2
spectro-scopy:seeing the ghosts of molecules
Kirchhoff ’s spectroscope, 1859
The Electromagnetic Spectrum
3
Spectrophotometry
Light source
• Tungsten lamp –VIS, NIR• Electric arc lamp (Xe, Hg, D2) – UV-VIS
Monochromator
• Prism• Grating (Czerny-Turner)
Aperture (slit) - spectral bandwidth
Cuvette
• optical glass, plastic –VIS• quartz, fused silica – UV
Detector
• Photoelements (diode, resistor) – UV-NIR• Photomultiplier tube (PMT) – UV-VIS• CCD array – UV-NIR• HgCdTe (MCT) – IR
single-beam spectrophotometer
Timescales of Biological Processes
1015 seconds = 31.7 million years
Time-Resolved Spectroscopy
• A very short laser pulse perturbs the system• The system is in non-equilibrium state• The time evolution of the optical properties is followed afterwards
Principle
• Fast and ultrafast processes – excited-state reactions, etc.• Temporal resolution of femtoseconds (10-15 s)• Temporal and spectral resolution tradeoff (Fourier-transform limit)
Information
• Short-lived intermediate reaction states• Transient concentrations• Reaction rate constants
Kinetic profile
6
Interactions of EM radiation with Matter
UV/VIS spectroscopy probes electronic excited states – electronic spectroscopy
IR spectroscopy probes molecular vibrations – vibrational spectroscopy
Absorption Emission Reflection
7
Molecular energy
Etotal = Evibrational + Eelectronic
E
S0-0
0-10-20-3
S1-0
1-11-21-3
S0-X – electronic ground stateS1-X – electronic excited stateSX-0 – vibrational ground stateSX-1 – vibrational excited state
8
Molecules exist in stationary states (eigenstates) with defined electronic nuclear and electronic configuration
The eigenstates are solutions of the Schrodinger equation
퐸Ψ = 퐻Ψ
The eigenstate with the lowest energy is the ground state.
The electronic wave functions correspond to molecular orbitals.
Absorption of Light
Absorption of UV/VIS light is a molecular transition between two different electronic levels.
9
• Electronic transition – between different electronic levels
• Vibrational transition – between different vibrational levels
• Vibronic transition – between different vibrational and electronic levels
• Absorption of UV/VIS light is an electronic or vibronic transition
hυ
S0-0
0-10-20-3
S1-0
1-11-21-3
Molecular Transitions - Perrin-Jablonski Diagram
Transition types:
Non-radiative
• Internal conversion
• Vibrational relaxation
• Intersystem crossing
• Quenching
Radiative
• Fluorescence
• Phosphorescence
• Delayed fluorescence
10
kic, kr, knr, … - rate constants
Rate Constants, Lifetimes, and Yields
Excited-state lifetime
휏 =1
푘 + 푘 + 푘
Quantum yield of fluorescence
휑 =푁푁
휑 =푘
푘 + 푘 + 푘
휑 =휏휏
휏 is the radiative lifetime
휏 =1푘
kr knr
kisc
Fluorescence Quenching
Any process that leads to quenching of the fluroescence
Dynamic quenching
Static quenching
Stern-Volmer equation퐹퐹 = 1 + 퐾 [푄]
F* Q F Q+ +
excited flurophore
quencher fluorophorein ground state
energy transfer
F* Q F Q+
flurophore quencher quencher complex
[ ][Q]
F 0/F
KSV
Förster Resonance Energy Transfer
Rate of energy transfer
푘 =9휅 푐
8휋휏 ∗푛 푅퐹 휔 휎 휔
d휔휔
�
�
Decreases with the sixth power of the distance
Is proportional to the overlap of the donor fluorescence spectrum and acceptor absorption spectrum
Depends on the mutual orientation of the donor and acceptor 휅 = 훍 훍 − 3(퐑 훍 )(퐑 훍 )
+ → +
A* B A B*S0
S1
13
Transient Absorption Spectroscopy
• ‘Pump’ pulse excites the system• A subsequent ‘probe’ pulse measures the changes induced by the pump
Δ퐴 휆, 푡 = 퐴 − 퐴 • 3rd-order nonlinear response• Sample interacts twice with the pump and once with the probe
푘 = 푘 − 푘 + 푘 = 푘
14Lasers in Medicine and Life Science, Szeged 2017
GS
S1
S2
pum
p
prob
e
Transient Absorption Spectra
GSB - ground-state bleaching
SE - stimulated emission
ESA, IA - excited-state absorption, induced absorption
15
M Vengris. Introduction to time-resolved spectroscopy
Lasers in Medicine and Life Science, Szeged 2017
Pump-Probe Measurement
16
Time-Resolved Fluorescence
Steady-state fluorescence intensity:
퐹 = 휉 퐼 휑
= 휉 퐼 1 − 푇 휑
Time-resolved fluorescence:
퐹 푡 = 퐴 푒
휏 =1
푘 + 푘 + 푘 +⋯
휑 =휏휏
• High sensitivity: 1000x more than traditional Abs
• High selectivity: single molecule in a living cell
• Information about excited-state dynamics
Advantages of fluorescence
• Absolute-valued units• Can distinguish yield and concentration• Resistant to optical artefacts
Advantages of time-resolved fluorescence
F
t
A
τf
Information from lifetime measurements
Fluorophore environment
Multiple conformations, conformational changes
Multiple environments
Interactions with neighbouring residues
Solvent relaxation
Fluorescence lifetime sensors (Ca2+, Mg2+)
Resonance energy transferLakowicz J.R. (2006) Springer
TRF quenching
TRF can distinguish between
• dynamic quenching (collisional quenching) – lifetime decrease with quencher concentration
• static quenching (exciplex formation) – lifetime is unchanged, amplitude decreases
TRF can distinguish different quenched populations
Lakowicz J.R. (2006) Springer
TRF spectroscopy – wavelength dependence
Global analysis of the kinetics at different emission wavelengths
• Components with closely spaced lifetimes
• Vast improvement in number of resolved lifetimes
Time-dependent spectral shifts
• Solvent relaxation dynamics
• General spectral evolution
Lakowicz J.R. (2006) Springer
Resolving multiple components
A* B*kAB = 5 ns-1
A B
0.5 ns-1 0.5 ns-1
0 0.5 1 1.5 2Time (ns)
0
0.2
0.4
0.6
0.8
1A*B*
푑퐴∗
푑푡 = − 푘 + 푘 퐴∗
푑퐵∗
푑푡 = 푘 퐴∗ − 푘 퐵∗
퐴∗ 푡 = 퐴 푒 .
퐵∗ 푡 = 1 − 퐴 푒 . + 퐴 푒 .
퐴∗ 푡 = 푎 푒 / + 푎 푒 /
퐵∗ 푡 = 푏 푒 / + 푏 푒 /
Decay-associated emission spectra
A* B*kAB = 5 ns-1
A B
0.5 ns-1 0.5 ns-1
퐹 휆, 푡 = 푎 (휆)푒 /�
DAS = 푎 (휆)
Methodology for TRF spectroscopy
Direct
Gating
Frequency-domain (CW)
Phase modulation
Time-domain (pulsed)
TCSPC Streak camera Upconversion
TCSPC is the most versatile and commonly used technique
Can resolve lifetimes from few ps to μs High dynamic range and signal-to-noise ratio
Time-Correlated Single-Photon Counting
CFD
ADC
Memory
Detector
Reference pulsesfrom light source
Histogram
threshold
zero cross
CFD
threshold
zero cross
TAC
stop
start
Range
Gain
Offset
AddressAMP
data+1
Adder
(time)Preamplifier
= control elementsSingle-photonpulses
Time-to-amplitude conversion:1. The laser pulse starts a clock 2. The detected fluorescence photon stops the clock3. The time between the Start and Stop signals is recorded
4. After many single photon events a histogram of decay times is collected
5. This histogram is the fluorescence decay kinetics
Original Waveform
Detector
Period 1
Period 5Period 6Period 7Period 8Period 9Period 10
Period N
Period 2Period 3Period 4
Resultafter
Photons
TimeSignal:
many
(Distribution of photon probability)
Time-correlated single-photon counting
Instrumentation for TCSPC: pulsed laser sources
Syncronously-pumped mode-locked dye lasers Ti:sapphire oscillators Diode lasers Fiber lasers
< 10 ps < 200 fs 50-70 ps < 10 ps
Expensive Expensive Inexpensive Inexpensive
Large footprint Large footprint Small footprint Small footprint
Vibration-sensitive Vibration-sensitive Vibration-tolerant Vibration-tolerant
Climate-sensitive Climate-sensitive Climate-tolerant Climate-tolerant
Difficult to align Hands-free alignment No alignment necessary No alignment necessary
Instrumentation for TCSPC: detection electronics
All electronics in a single board/module
Fully automatized
Affordable
Useful for both
• Spectroscopy (TCSPC) • Microscopy (FLIM)
Becker & Hickl SPC-1x0TCSPC board
PicoQuant PicoHarp 300Stand-alone TCSPC module
Light Harvesting in Photosynthesis
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Primary photochemistry only takes place in reaction center pigments
Majority of pigments are part of light-harvesting antenna complexes
LHAs deliver absorbed light energy to RC via excitation energy transfer
Photoinduced Electron Transport
Time-Resolved Fluorescence of Plant Photosystem I
Mazor et al. (2015)
Electron transfer in PSI
Nelson & Yocum, Annu. Rev. Plant Biol., 2006, 57:521-565 31
Time-Resolved Fluorescence of Plant Photosystem I
PSI-LHCI PSI core
Akhtar et al. (2018) Photosynth. Res.
LHCI
PSI core
45 ns-1
(22 ps)
LHCI
0.45 ns-1
(2.2 ns)
17 ns-1
(58 ps)-1
11 ns-1
(90 ps)
Literature
1. Lakowicz J.R. Principles of Fluorescence Spectroscopy, 3rd ed., 2006, Springer
2. Lambrev P.H. & Garab G. Optical spectroscopy tools to investigate the molecular organization and function of photosynthetic protein complexes, In: Selected Topics from Contemporary Experimental Biology, Vol. 2, 2015, BRC, pp. 269-288
3. Garab G. & Van Amerongen H. Linear dichroism and circular dichroism in photosynthesis research, 2009, Photosynth. Res. 101:135-136
4. Mukamel S. Principles of Nonlinear Optical Spectroscopy, 1995, Oxford University Press
5. Andrews D.L. & Demidov A.A. An Introduction to Laser Spectroscopy, 2002, Springer