The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH/NH binding. Time-resolved detection of O formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NHCl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV) (S) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the "dangling" Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S-S transition as well as their reversal in the O formation transition, in line with the carousel mechanism [Askerka, M., et al. (2015) Biochemistry 54, 5783]. (5) We observe the same partial inhibition of PSII by NHCl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the results described above are valid for plant and cyanobacterial PSII.

We report on combined simultaneous temporal and spatial laser pulse shaping by utilizing light polarization properties. Thereto, a setup comprising a temporal pulse shaper, a waveplate, and a spatial shaper was developed and characterized by comparison with simulations. This enables to simultaneously shape one polarization component temporally and spatially while the perpendicular polarization component is modified temporally. The spatially and temporally modulated light fields were recorded and visualized by suitable contour plots, which was particularly demonstrated for cylindrically symmetric pulse profiles. Moreover, temporally and spatially shaped pulses were applied for two-photon excited fluorescence of dyes. These measurements were conducted by scanning third order phase functions for specific spatial pulse components which yields an enhanced contrast difference between fluorescing dyes. The presented temporal and spatial shaping method of ultrashort laser pulses has a high potential for biophotonic applications.

We report combined temporal and spatial laser pulse shaping to perform lateral and depth dependent two-photon excited fluorescence of dyes. For generating the specific spatially and temporally phase tailored pulses a temporal pulse shaper and a subsequent spatial pulse shaper are employed. Simultaneous spatial and temporal shaping is presented for two-photon excited fluorescence by applying temporal third order phase functions on spatially different light field components. Moreover, the prospects of spatial shaping are demonstrated by applying various lateral two-photon fluorescence pattern. In particular, a depth dependent excitation of different dyes is performed which leads to a high axially resolved fluorescence contrast. The introduced spatial and temporal shaping technique provides new perspectives for biophotonic imaging applications.

We present contrast enhancement for the autofluorescing coenzymes flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH) in glycerol using phase and polarization shaped laser pulses after the transmission through a kagome fiber. Thereto, we report a way to calculate the optimal light modulator incident polarization angle, which in general differs from the horizontal. Combining phase and polarization shaping, we can selectively excite FAD in one polarization and simultaneously NADH in the other polarization direction by third order phase functions. Due to high anisotropy, the contrast of the fluorescence depends on the polarization direction. The effect of the fiber on the phase is precompensated in order to obtain the desired phase function after the fiber. Since the relative amounts of NADH and FAD give information about cellular metabolic activity which in turn helps understand disease processes, the method promises high biophotonic potential.

In recent years fluorescent dyes were used as markers for imaging applications where ultrashort laser pulses were employed to distinguish between certain structures in biological samples. A large contrast is favorable in this regard to receive a clear microscopic image. In this context, the technique of laser pulse shaping provides a powerful tool to tailor the pulses such that two fluorescence dyes can be selectively excited [1,2]. Laser pulse shaping for control of photo-induced molecular processes has attained considerable success since it enables to drive the induced processes at a maximum yield along desired paths [3,4]. Moreover, a parametric subpulse encoding was developed [5], where physically intuitive pulse parameters like chirps and polarization states can be controlled which opens new perspectives of utilizing the light field in the pulse modulation. Pulse shaping techniques were already employed in life sciences in order to investigate biologically relevant systems. Here, laser pulse shaping is often applied to multiphoton excitation where intrapulse interference becomes relevant [6]. This enables to exploit interference effects in multiphoton excited fluorescence spectroscopy [7,8] and allows for three-dimensional imaging by multiphoton microscopy [9,10]. Moreover, this permits to steer molecular processes by utilizing these pulses for inducing specific multiphoton processes in molecular systems. However, in many cases the excitation spectra of the different substances are close to each other or even overlap which impedes a selective excitation. Furthermore, perturbing spectral features may hamper the intended excitations of the examined species. Methods that allow to bypass these unwanted transitions would be desirable in order to improve the received contrast. In this contribution phase, amplitude, and polarization pulse shaping methods are described to control different excitation processes. The tailored laser pulses cause selective multiphoton induced fluorescence of dye mixtures. Special scans of frequency-shifted antisymmetric phase functions will be employed to control the multiphoton excitation fluorescence [11]. The efficiency at which phase and amplitude modulated pulses with low amplitude ranges enhance imaging contrast is recorded, and the results are compared to calculations. Additionally, it will be demonstrated how a two-photon excitation can be tailored to bypass a simulated perturbing one-photon transition located in the same spectral region. In the last part, another way of circumvention by going over to the perpendicular polarization direction is realized by polarization shaping in order to increase the contrast between different dyes, whereby the phase-tailored two-photon excitation is addressed in one polarization direction and the one-photon transition is selectively excited in the other polarization direction. The presented method of phase, amplitude, and polarization pulse