Ultrafast transient absorption spectroscopy: principles and application to photosynthetic systems

Abstract

The photophysical and photochemical reactions, after light absorption by a photosynthetic pigment-protein complex, are among the fastest events in biology, taking place on timescales ranging from tens of femtoseconds to a few nanoseconds. The advent of ultrafast laser systems that produce pulses with femtosecond duration opened up a new area of research and enabled investigation of these photophysical and photochemical reactions in real time. Here, we provide a basic description of the ultrafast transient absorption technique, the laser and wavelength-conversion equipment, the transient absorption setup, and the collection of transient absorption data. Recent applications of ultrafast transient absorption spectroscopy on systems with increasing degree of complexity, from biomimetic light-harvesting systems to natural light-harvesting antennas, are presented. In particular, we will discuss, in this educational review, how a molecular understanding of the light-harvesting and photoprotective functions of carotenoids in photosynthesis is accomplished through the application of ultrafast transient absorption spectroscopy.

Fig. 1

Left panel: Schematic depiction of the transient absorption spectroscopy principle. Right panel: Contributions to a ΔA spectrum: ground-state bleach (dashed line), stimulated emission (dotted line), excited-state absorption (solid line), sum of these contributions (gray line)

Fig. 2

Schematic representation of an experimental ultrafast transient absorption setup

Fig. 3

a Molecular structure of a carotenophthalocyanine light-harvesting dyad 1. b Evolution-associated difference spectra (EADS) that result from a global analysis on transient absorption experiments on dyad 1. The excitation wavelength was 475 nm. c Kinetic traces at 560 nm (upper panel) and 680 nm (lower panel). d Kinetic scheme that describes the excited-state processes in dyad 1 upon carotenoid excitation. Solid lines denote energy transfer, dotted denote internal conversion, dashed denotes intersystem crossing processes. Source: Berera et al. (2007)

Fig. 4

a Molecular structure of a carotenophthalocyanine light-harvesting dyad 1, 2, and 3. The carotenoids of dyad 1, 2 and 3 contain 9, 10 and 11 conjugated C=C double bonds, respectively. b Upper panel: kinetic traces at 680 nm of dyad 1, 2, and 3 and a model Pc in tetrahydrofuran (THF). Lower panel: kinetic traces of dyad 3 dissolved in acetone detected at 480 nm (solid line) and 576 nm (dashed line). Excitation wavelength for b and d was 680 nm. c Kinetic scheme that describes the excited-state decay processes In dyad 2 and 3 upon Pc excitation. Solid line denotes energy transfer, dotted line denotes internal conversion process. d Evolution-associated difference spectra (EADS) that result from a global analysis on transient absorption experiments on dyad 3 dissolved in acetone. Source: Berera et al. (2006)

Fig. 5

Selected kinetic traces for unquenched LHCII trimers (a) and quenched LHCII aggregates (b) at 677 nm (top), 489 nm (middle) and 537 nm (bottom), following a 100 fs, 10 nJ laser pulse at 675 nm. The vertical axis shows the measured change in absorption, the horizontal axis is linear up to 1 ps and logarithmic thereafter. The long short-dashed line represents the 1 ps phase due to chlorophyll excited state relaxation, the dotted line the excited state decay of chlorophyll, the dashed line the absorption changes due to the quencher Q, and the dash-dotted line the build-up of the triplet state. The kinetic model is shown in (c) and the corresponding species-associated difference spectra (SADS) in (d). Source: Ruban et al. (2007)