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

28

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