Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.

Here we report on a set of programs developed at the ZMBH Bio-Imaging Facility for tracking real-life images of cellular processes. These programs perform 1) automated tracking; 2) quantitative and comparative track analyses of different images in different groups; 3) different interactive visualization schemes; and 4) interactive realistic simulation of different cellular processes for validation and optimal problem-specific adjustment of image acquisition parameters (tradeoff between speed, resolution, and quality with feedback from the very final results). The collection of programs is primarily developed for the common bio-image analysis software ImageJ (as a single Java Plugin). Some programs are also available in other languages (C++ and Javascript) and may be run simply with a web-browser; even on a low-end Tablet or Smartphone. The programs are available at https://github.com/nurlicht/CellulyzerDemo

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

A modulated icosahedral shell with an inclusion is a concise description of many viruses, including recently-discovered large double-stranded DNA ones. Many X-ray scattering patterns of such viruses show major polygonal fringes, which can be reproduced in image reconstruction with a homogeneous icosahedral shell. A key question regarding a low-resolution reconstruction is how to introduce further changes to the 3D profile in an efficient way with only a few parameters. Here, we derive and compile different analytical models of such an object with consideration of practical optical setups and typical structures of such viruses. The benefits of such models include 1) inherent filtering and suppressing different numerical errors of a discrete grid, 2) providing a concise and meaningful set of descriptors for feature extraction in high-throughput classification/sorting and higher-resolution cumulative reconstructions, 3) disentangling (physical) resolution from (numerical) discretization step and having a vector graphics format for visualization or further analysis at arbitrary scales, 4) eliminating the phase-retrieval step and enforcing transparent, relevant, and controlled type/level of a-priori information in a real-space formulation, and 5) evaluating the reflections and surface resonances of an icosahedral object, and hence corrections for the common scattering model.

X-ray scattering patterns from emerging single particle experiments have commonly many missing or contaminated pixels. This complicates different analyses including projections on Fourier or other basis functions (for noise suppression, compression, feature extraction, or retrieval of real-space patterns), as they require integration over all pixels. Here, we derive alternative formulations for Discrete Fourier Transform and a common orthogonal basis by explicit consideration of missing pixels and finite size. Such linear formulations exclude the nonlinear distortion that would be caused by multiplication of the complete scattering pattern with the mask function. Contrary to nonlinear and non-convex phase retrieval optimizations, such reduced-dimension formulations can be used to fully enforce the constraints and to retrieve unknown intensities in a linear fashion. Applications are demonstrated for some typical cases, and extensions to more general cases are discussed.

Emerging coherent X-ray scattering patterns of single-particles have shown dominant morphological signatures in agreement with predictions of the scattering model used for conventional protein crystallography. The key question is if and to what extent these scattering patterns contain volumetric information, and what model can retrieve it. This contribution is Part 2 out of two reports, in which we seek to clarify the assumptions of some different regimes and models of X-ray scattering and their implications for single particle imaging. In Part 1, basic concepts and existing scattering models along with their implications for nanocrystals, and also the misconception of using Diffraction Theory for volumetric scattering were addressed. Here in Part 2, specific challenges ahead of single particle imaging are addressed. Limitations of the conventional scattering model in the test case of a sphere, schemes for improving this common scattering model, ambiguities in meaning and properties of "density map" and non-classical effects, the crucial role of electromagnetic boundary conditions, the uniqueness of X-ray scattering inverse problems, and additional vulnerabilities of phase retrieval and its relation with "resolution" are discussed. We raise concerns about the unverified use of the common scattering model of protein crystallography for arbitrary objects, and also leaving fundamental questions such as meaning/uniqueness/properties of the sought 3D profiles to phase retrieval algorithms.

Single particle 3D imaging with ultrashort X-ray laser pulses is based on collecting and combining the information content of 2D scattering patterns of an object at different orientations. Typical sample-delivery schemes leave little or no room for controlling the orientations. As such, the orientation associated with a given snapshot should be estimated after the experiment. Here we present an open-source code for the most rigorous technique having been reported in this context. Some practical issues along with proposed solutions are also discussed.

Coherent diffractive imaging with x-ray free-electron lasers (XFEL) promises high-resolution structure determination of noncrystalline objects. Randomly oriented particles are exposed to XFEL pulses for acquisition of two-dimensional (2D) diffraction snapshots. The knowledge of their orientations enables 3D imaging by multiview reconstruction, combining 2D diffraction snapshots in different orientations. Here we introduce a globally optimal algorithm that can infer these orientations. We apply it to experimental XFEL data of nanoparticles and so determine their 3D electron density.

We explore strategies for optimizing selectivity, specificity, and sensitivity in broadband CARS by precalculating pulse shapes using an evolutionary algorithm. We show the possibility of selective excitation of a single constituent in a test case of a mixture of five resonant compounds. The obtainable contrast ratio for a test case of PMMA in a mixture of five resonant compounds is predicted to be 2000:1, and is related the uniqueness of the complex vibrational response of the compound of interest compared to that of the surrounding molecules. Furthermore we investigate how the effects of homodyne mixing in the focal volume affect the obtainable contrast ratio and how noise affects the optimization. We also show preliminary results of experimental optimization of the CARS signal from PMMA microspheres, resulting in high contrast imaging, free of non-resonant background signal.

Identifying complex molecules often entails detection of multiple vibrational resonances, especially in the case of mixtures. Phase shaping of broadband pump and probe pulses allows for the coherent superposition of several resonances, such that specific molecules can be detected directly and with high selectivity. Our particular implementation of coherent anti-Stokes Raman scattering (CARS) spectroscopy and imaging employs broadband pump and probe fields in combination with a narrowband Stokes field. We describe our approach for combining spectral phase shaping and closed-loop optimization strategies to perform chemically-selective microscopy. To predict the optimal excitation profile we employ evolutionary algorithms that use the vibrational phase responses of five distinct molecules with overlapping resonances and investigate the effect of phase instability on the optimization. We have recently shown that modified polynomials and orthogonal rational functions can give rise to improved contours for CARS fitness landscapes. Now, by considering the landscapes associated with different basis sets, we introduce two figures of merit to quantitatively rank basis functions in terms of their "appropriateness" for modeling nonlinear phase-shaped processes.

We investigate the benefits of estimating the group delay spectrum (as opposed to the phase spectrum) on the performance of an adaptive laser pulse shaping experiment. We use both numerical and experimental approaches, and consider different figures of merit such as efficiency, noise robustness, convergence speed, repeatability, fitness sensitivity, and parameter insensitivity. While primarily focused on the process of broadband second harmonic generation, we also study a resonant third order nonlinear process. Our results show an overall superiority of delay-based formulation in the cases under study (compared to a conventional phase-based formulation) for adaptive laser pulse shaping.

Structural and optical characterization of water soluble, thermo-responsive quantum dot/poly(N-isopropyl acrylamide) (QD/PNIPAM) hybrid particles using fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCSPC) measurements performed at temperatures below and above the lower critical solution temperature (LCST) of PNIPAM is reported. By increasing the temperature above the LCST, the signature of the PNIPAM chain collapse covering the QDs is revealed by FCS measurements. Despite the significant structural change, the TCSPC measurements show that the fluorescence lifetimes remain of the same order of magnitude at T > LCST. Such QD/PNIPAM hybrid particles with water solubility and robust thermo-responsive behavior at physiologically relevant temperatures are potentially useful for (bio)molecular sensing and separation applications.

The identification of large molecules in complex environments requires probing of multiple vibrational resonances rather than a single resonance. Phase-shaping the excitation pulses allows the coherent mixing of several resonances so that the presence of molecules can be inferred directly from the integrated output pulse energy. This avoids the need for the collection of spectra or multiple measurements. This article describes a particular implementation for coherent anti-Stokes Raman scattering microscopy that uses a broadband pump and probe field in combination with a narrowband Stokes field. We numerically study the possibilities of optimizing selectivity, specificity, and sensitivity by precalculating pulse shapes using an evolutionary algorithm. Copyright c � 2011 John Wiley & Sons, Ltd.

Pulse shaping techniques are used to improve the selectivity of broadband CARS experiments, and to reject the overwhelming background.Knowledge about the fitness landscape and the capability of tailoring it is crucial for both fundamental insight and performing an efficient optimization of phase shapes.We use an evolutionary algorithm to find the optimal spectral phase of the broadband pump and probe beams in a background-suppressed shaped CARS process.We then investigate the shapes, symmetries, and topologies of the landscape contour lines around the optimal solution and also around the point corresponding to zero phase.We demonstrate the significance of the employed phase bases in achieving convex contour lines, suppressed local optima, and high optimization fitness with a few (and even a single) optimization parameter.

Finding an optimal phase pattern in a multidimensional solution landscape becomes easier and faster if local optima are suppressed and contour lines are tailored towards closed convex patterns. Using wideband second harmonic generation as a coherent control test case, we show that a linear combination of spectral phase basis functions can result in such improvements and also in separable phase terms, each of which can be found independently. The improved shapes are attributed to a suppressed nonlinear shear, changing the relative orientation of contour lines. The first order approximation of the process shows a simple relation between input and output phase profiles, useful for pulse shaping at ultraviolet wavelengths.

Many spectroscopic applications of femtosecond laser pulses require properly-shaped spectral phase profiles. The optimal phase profile can be programmed on the pulse by adaptive pulse shaping. A promising optimization algorithm for such adaptive experiments is evolution strategy (ES). Here, we report a four fold increase in the rate of convergence and ten percent increase in the final yield of the optimization, compared to the direct parameterization approach, by using a new version of ES in combination with Legendre polynomials and frequency-resolved detection. Such a fast learning rate is of paramount importance in spectroscopy for reducing the artifacts of laser drift, optical degradation, and precipitation.

Ultrafast relaxation of a carotenoid in an artificial light-harvesting complex has been studied by transient absorption spectroscopy. The transient signal amplitudes at several wavelengths as well as the amplitudes of the underlying species associated spectra (SAS) are analysed for several excitation energies ranging over more than two orders of magnitude (10nJ/pulse up to 3000nJ/pulse). Our analysis shows that the contribution from the so-called S∗ signal on the long-wavelength side of the first allowed S0→S2 transition has a markedly different excitation energy dependence and saturation behaviour than the electronic excited state S1. These observations are modelled and explained in terms of a two-photon excitation of a vibrationally hot ground state via an impulsive stimulated Raman scattering (ISRS). The experimental observations of the varying pulse energy dependencies of different excited state species are supported by an analysis based on a density-matrix formalism.

Photodynamic therapy (PDT) is a treatment based on the interaction of light, photosensitizing agents and tissue oxygen. The light delivery in PDT is usually optimized by controlling the intensity, the spectrum, and/or the dosage of excitation light. In this paper, we introduce a novel method that aims to improve the efficiency of PDT by controlling the phase of the excitation light, an important and so far neglected parameter. This coherent control approach utilizes the coherence properties of light-matter interaction and aims to manipulate the quantum interferences between various available reaction pathways. In general, an outcome of a photochemical reaction can be optimized by enhancing the desired reaction pathways and suppressing other unwanted pathways. Such optimizations can be done by appropriate tailoring of the electric field profile of a broadband coherent excitation light, i.e. ultrafast laser pulse. Here, we used a femtosecond laser source with adaptive pulse shaping together with a molecular feedback in a learning loop to search for and synthesize such 'smart' laser pulses. Our control objective is to enhance the triplet yield of a model photosensitizer zinc phthalocyanine (ZnPc), which then leads to enhancement of the overall PDT process. We use two coherent control schemes where we optimize the ratio between the excited singlet state (S) and triplet state (T) ZnPc molecules both ways (S/T and T/S). We demonstrate a control of 15% over the triplet yield between the found best and the worst pulse shapes. Our preliminary results show that phase shaping can indeed be used in manipulating photosensitizer photophysics and correspondingly the yield of singlet oxygen.