The wild type green fluorescent protein (GFP) from Aequorea victoria has been extensively investigated with a strong focus on the photochemistry and structural dynamics that are linked with its diverse activities. GFP combines a number of remarkable, and some unique, features that are still intensely researched both experimentally and theoretically. The protein environment effectively inhibits deactivation pathways that are dominant in the isolated chromophore and is therefore responsible for the bright fluorescence. Its p-hydroxybenzylidene-imidazolidinone chromophore acts as a photoacid, and optical excitation triggers ultrafast proton transfer reactions in the active site. The microscopic details of the proton transfer mechanism through a hydrogen bonding network are discussed in this critical review. This property of the wild type GFP has provided the opportunity to characterise the role of the specific protein environment in the proton transfer reactions in comparison to photoacid reactions in the condensed phase. In addition, GFP displays a photochromic side reaction that is uniquely caused by electron transfer from a buried anionic glutamic acid to the optically excited chromophore. This phototransformation property has also been exploited in high resolution fluorescence microscopy techniques. The discussion in this review is extended to include vibrational spectroscopy and structural dynamics (106 references).

Current advanced laser, optics and electronics technology allows sensitive recording of molecular dynamics, from single resonance to multi-colour and multi-pulse experiments. Extracting the occurring (bio-) physical relevant pathways via global analysis of experimental data requires a systematic investigation of connectivity schemes. Here we present a Matlab-based toolbox for this purpose. The toolbox has a graphical user interface which facilitates the application of different reaction models to the data to generate the coupled differential equations. Any time-dependent dataset can be analysed to extract time-independent correlations of the observables by using gradient or direct search methods. Specific capabilities (i.e. chirp and instrument response function) for the analysis of ultrafast pump-probe spectroscopic data are included. The inclusion of an extra pulse that interacts with a transient phase can help to disentangle complex interdependent pathways. The modelling of pathways is therefore extended by new theory (which is included in the toolbox) that describes the finite bleach (orientation) effect of single and multiple intense polarised femtosecond pulses on an ensemble of randomly oriented particles in the presence of population decay. For instance, the generally assumed flat-top multimode beam profile is adapted to a more realistic Gaussian shape, exposing the need for several corrections for accurate anisotropy measurements. In addition, the (selective) excitation (photoselection) and anisotropy of populations that interact with single or multiple intense polarised laser pulses is demonstrated as function of power density and beam profile. Using example values of real world experiments it is calculated to what extent this effectively orients the ensemble of particles. Finally, the implementation includes the interaction with multiple pulses in addition to depth averaging in optically dense samples. In summary, we show that mathematical modelling is essential to model and resolve the details of physical behaviour of populations in ultrafast spectroscopy such as pump-probe, pump-dump-probe and pump-repump-probe experiments.

Photosystem I (PSI) from Chroococcidiopsis thermalis PCC 7203 grown under far-red light (FRL; >725 nm) contains both chlorophyll a and a small proportion of chlorophyll f. Here, we investigated excitation energy transfer and charge separation using this FRL-grown form of PSI (FRL-PSI). We compared femtosecond transient visible absorption changes of normal, white-light (WL)-grown PSI (WL-PSI) with those of FRL-PSI using excitation at 670 nm, 700 nm, and (in the case of FRL-PSI) 740 nm. The possibility that chlorophyll f participates in energy transfer or charge separation is discussed on the basis of spectral assignments. With selective pumping of chlorophyll f at 740 nm, we observe a final ∼150 ps decay assigned to trapping by charge separation, and the amplitude of the resulting P700+•A1-• charge-separated state indicates that the yield is directly comparable to that of WL-PSI. The kinetics shows a rapid 2 ps time constant for almost complete transfer to chlorophyll f if chlorophyll a is pumped with a wavelength of 670 nm or 700 nm. Although the physical role of chlorophyll f is best supported as a low-energy radiative trap, the physical location should be close to or potentially within the charge-separating pigments to allow efficient transfer for charge separation on the 150 ps timescale. Target models can be developed that include a branching in the formation of the charge separation for either WL-PSI or FRL-PSI.

The quantum yield of photoisomerisation of the photoactive yellow protein (PYP) strongly depends on peak power and wavelength with femtosecond optical excitation. Using systematic power titrations and addition of second order dispersion resulting in 140, 300 and 600 fs pulse durations, the one and multi-photon cross-sections at 400, 450 and 490 nm have been assessed from transient absorption spectroscopy and additionally the Z-scan technique. Applying a target model that incorporates photoselection theory, estimates for the cross-sections for stimulated emission and absorption of the first excited state, the amount of ultrafast internal conversion and the underlying species associated dynamics have been determined. The final quantum yields for photoisomerisation were found to be 0.06, 0.14–0.19 and 0.02 for excitation wavelengths 400, 450 and 490 nm and found to increase with increasing pulse durations. Transient absorption measurements and Z-scan measurements at 450 nm, coinciding with the maximum wavelength of the ground state absorption, indicate that the photochemical quantum yield is intrinsically limited by an ultrafast internal conversion reaction as well as by stimulated emission cross-section. With excitation at 400 nm photoisomerisation quantum yield is further significantly limited by competing multi-photon excitation into excited state absorption at 385 nm previously proposed to result in photoionisation. With excitation at 490 nm the photoisomerisation quantum yield is predominantly limited further by the significantly higher stimulated emission cross-section compared to ground state cross-section as well as multi-photon processes. In addition to photoionisation, a second product of multi-photon excitation is identified and characterised by an induced absorption at 500 nm and a time constant of 2 ps for relaxation. With power densities up to 138 GW cm⁻² the measurements have not provided indication for coherent multi-photon absorption of PYP. In the saturation regime with 450 nm excitation, the limit for the photoisomerisation quantum yield was found to be 0.14–0.19 and the excited state absorption cross-section 6.1 × 10⁻¹⁷ cm² or 0.36 times the ground state cross-section of 1.68 × 10⁻¹⁶ cm² per molecule. This places a fundamental restriction on the maximum populations and sample penetration that may be achieved for instance in femtosecond pump–probe experiments with molecular crystals of PYP.