P. Lambrev October 10, 2018

Transcription

1 TIME-RESOLVED OPTICAL SPECTROSCOPY Petar Lambrev Laboratory of Photosynthetic Membranes Institute of Plant Biology The Essence of Spectroscopy spectro-scopy: seeing the ghosts of molecules Kirchhoff s spectroscope, The Electromagnetic Spectrum 3 1

2 Spectrophotometry single-beam spectrophotometer Light source Tungsten lamp VIS, NIR Electric arc lamp (Xe, Hg, D 2) 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 Timescales of Biological Processes seconds = 31.7 million years Time-Resolved Spectroscopy Principle 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 Information Fast and ultrafast processes excited-state reactions, etc. Temporal resolution of femtoseconds (10-15 s) Temporal and spectral resolution tradeoff (Fourier-transform limit) Kinetic profile Short-lived intermediate reaction states Transient concentrations Reaction rate constants 6 2

3 Interactions of EM radiation with Matter Absorption Emission Reflection UV/VIS spectroscopy probes electronic excited states electronic spectroscopy IR spectroscopy probes molecular vibrations vibrational spectroscopy 7 Molecular energy E total = E vibrational + E electronic E S S 0-0 S 0-X electronic ground state S 1-X electronic excited state S X-0 vibrational ground state S X-1 vibrational excited state Molecules exist in stationary states (eigenstates) with defined electronic nuclear and electronic configuration The eigenstates are solutions of the Schrodinger equation EΨ = H Ψ The eigenstate with the lowest energy is the ground state. The electronic wave functions correspond to molecular orbitals. 8 Absorption of Light Absorption of UV/VIS light is a molecular transition between two different electronic levels. hυ S S 0-0 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 9 3

4 Molecular Transitions - Perrin-Jablonski Diagram Transition types: Non-radiative Internal conversion Vibrational relaxation Intersystem crossing Quenching Radiative Fluorescence Phosphorescence Delayed fluorescence k ic, k r, k nr, - rate constants 10 Rate Constants, Lifetimes, and Yields k isc Excited-state lifetime 1 τ = k + k + k Quantum yield of fluorescence φ = N N k r k nr k φ = k + k + k φ = τ τ τ is the radiative lifetime τ = 1 k Fluorescence Quenching Any process that leads to quenching of the fluroescence Dynamic quenching energy transfer Stern-Volmer equation F F = 1 + K [Q] F* + Q F + Q excited flurophore quencher fluorophore in ground state Static quenching F 0 /F F* + Q [ F Q ] flurophore quencher quencher complex K SV [Q] 4

5 P. Lambrev October 10, 2018 Förster Resonance Energy Transfer S S 0 A* B A B* Rate of energy transfer k = 9κ c 8πτ n R F ω σ ω dω ω 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 κ = μ μ 3(R μ )(R μ ) 13 Transient Absorption Spectroscopy S2 S1 pump probe GS Pump pulse excites the system A subsequent probe pulse measures the changes induced by the pump ΔA λ, t = A A 3 rd -order nonlinear response Sample interacts twice with the pump and once with the probe k = k k + k = k Lasers in Medicine and Life Science, Szeged Transient Absorption Spectra GSB - ground-state bleaching SE - stimulated emission ESA, IA - excited-state absorption, induced absorption M Vengris. Introduction to time-resolved spectroscopy Lasers in Medicine and Life Science, Szeged

6 Pump-Probe Measurement 16 Time-Resolved Fluorescence Advantages of fluorescence Steady-state fluorescence intensity: F = ξ I φ High sensitivity: 1000x more than traditional Abs High selectivity: single molecule in a living cell Information about excited-state dynamics = ξ I 1 T φ Time-resolved fluorescence: F t = A e 1 τ = k + k + k + Advantages of timeresolved fluorescence F φ = τ τ Absolute-valued units Can distinguish yield and concentration Resistant to optical artefacts A τ f t Information from lifetime measurements Fluorophore environment Multiple conformations, conformational changes Multiple environments Interactions with neighbouring residues Solvent relaxation Fluorescence lifetime sensors (Ca 2+, Mg 2+ ) Resonance energy transfer Lakowicz J.R. (2006) Springer 6

7 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 k AB = 5 ns A* -1 B* A 0.5 ns ns -1 B Population da dt = k + k A db dt = k A k B A t = a e / + a e / B t = b e / + b e / A t = A e. B t = 1 A e. + A e. 7

8 P. Lambrev October 10, 2018 Decay-associated emission spectra F λ, t = a (λ)e / DAS = a (λ) k AB = 5 ns A* -1 B* 0.5 ns ns -1 A B Methodology for TRF spectroscopy Direct Frequencydomain (CW) Time-domain (pulsed) Gating Phase modulation 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 threshold Reference pulses from light source Range CFD start zero cross Gain Mem ory Histogram threshold TAC AMP ADC Address (tim e) Pream plifier stop Detector Singlephoton CFD zero cross Offset = control elements data +1 Adder pulses Time-to-amplitude conversion: 1. The laser pulse starts a clock 2. The detected fluorescence photon stops the clock 3. The time between the Start and Stop signals is recorded 8

9 Time-correlated single-photon counting Detector Signal: Period 1 Period 2 Period 3 Period 4 Period 5 Period 6 Period 7 Period 8 Period 9 Period 10 Original Waveform (Distribution of photon probability) Time 4. After many single photon events a histogram of decay times is collected 5. This histogram is the fluorescence decay kinetics Period N Result after many Photons Instrumentation for TCSPC: pulsed laser sources Syncronously-pumped mode-locked dye lasers Ti:sapphire oscillators Diode lasers Fiber lasers < 10 ps < 200 fs 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-1x0 TCSPC board PicoQuant PicoHarp 300 Stand-alone TCSPC module 9

10 Light Harvesting in Photosynthesis 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 28 Photoinduced Electron Transport Time-Resolved Fluorescence of Plant Photosystem I Mazor et al. (2015) 10

11 Electron transfer in PSI Nelson & Yocum, Annu. Rev. Plant Biol., 2006, 57: Time-Resolved Fluorescence of Plant Photosystem I PSI-LHCI PSI core LHCI 0.45 ns -1 (2.2 ns) LHCI 17 ns -1 (58 ps) ns -1 (90 ps) PSI core 45 ns -1 (22 ps) Akhtar et al. (2018) Photosynth. Res. Literature 1. Lakowicz J.R. Principles of Fluorescence Spectroscopy, 3 rd 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 Garab G. & Van Amerongen H. Linear dichroism and circular dichroism in photosynthesis research, 2009, Photosynth. Res. 101: 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 11