Search

Imaging spectroscopy has been developed in the last two decades in the visible and infrared spectral range for detecting pigments and binders on paintings. The near-infrared (NIR) region has been proved effective for the discrimination of lipids and proteinaceous binders. More recently, the mid-infrared (MIR) range has also been tested on paintings. Reflection imaging prototypes already developed could be further optimized for cultural heritage analysis, for example by: enhancing the instrument configuration and performance; adopting compressive strategies to increase data processing speeds; using data validation to confirm that the processed image reflects the composition of a painted surface; and lowering price to enable more cost-effective analysis of large surface areas. Here, we demonstrate a novel hyperspectral Fourier transform spectrometer (HS FTS), which enables an imaging strategy that provides a significant improvement in acquisition rate compared to other state-of-the-art techniques. We demonstrate hyperspectral imaging across the 1400–700 cm−1 region in reflection mode with test samples and the painting ‘Uplands in Lorne’ (Acc. No.: GLAHA43427) by D.Y. Cameron (1865–1945). A post-processing analysis of the resulting hyperspectral images, after validation of reference samples by conventional Fourier transform infrared spectroscopy, shows the potential of the method for efficient non-destructive classification of different materials found on painted cultural heritage. This research demonstrates that the HS FTS is a convenient and compact tool for non-invasive analysis of painted cultural heritage objects at spatio-spectral acquisition rates potentially higher than current FTS imaging techniques. Ultimately, when combined with fast graphics processing unit-based reconstruction, the HS FTS may enable fast, large area imaging. Graphical abstract

The discovery1,2and the pioneering applications3of CRISPR-Cas effector complexes have provided powerful gene-editing tools. The effector complexes are guided to the targeted genomic locus by the complementarity of their CRISPR RNA (crRNA)4,5. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base-pairing between crRNA and the DNA target strand resulting in the formation of an R-loop structure5,6. Full R-loop formation is the prerequisite for the subsequent DNA cleavage. While the CRISPR-Cas technology is easy to use, efficient and highly versatile, therapeutic applications are hampered by the off-target effects due to the recognition of unintended sequences with multiple mismatches7. This process is still poorly understood on a mechanistic level8,9. Particularly, the lack of insight into the energetics and dynamics of the R-loop formation hinders a direct modelling of the R-loop formation for off-target prediction.Here we set up ultrafast DNA unwinding experiments based on plasmonic DNA nanorotors to follow the R-loop formation by the Cascade effector complex in real time, close to base pair resolution. We directly resolve a weak global downhill bias of the energy landscape of the forming R-loop followed by a steep uphill bias for the final base pairs. We furthermore show a modulation of the landscape by base flips and mismatches. These data provide that Cascade-mediated R-loop formation occurs on short time scales in single base pair steps of sub-millisecond duration, but on longer time scales in six–base pair intermediate steps in agreement with the structural periodicity of the crRNA-DNA hybrid. We expect that the knowledge about the energy landscapes of R-loop formation of CRISPR-Cas effector complexes will pave the way for a detailed understanding and prediction of off-target recognition10.

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-pairing between the crRNA of the complexes and the DNA target strand such that an R-loop is formed. R-loop formation starts at a specific PAM motif and progresses reversibly in single base-pair steps until mismatches stop further progression or until the full target is recognized and destroyed. The reversible nature of this process entails that even a fully matching target should only become recognized with a low probability per target encounter. The details of this process, which directly affect the effectiveness of the target search, remain unresolved.Here we dissect the target search process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the low target recognition probabilities and show that they increase with increasing negative supercoiling. Furthermore, we demonstrate that Cascade uses a combination of three-dimensional and limited one-dimensional diffusion along the DNA contour for its target search. The latter allows for rapidly scanning the PAM sequences in a given region and, importantly, significantly increasing the overall efficiency of the target search by repeatedly revisiting the sites. Overall we show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

Spectrally-resolved imaging provides a spectrum for each pixel of an image that, in the mid-infrared, can enable its chemical composition to be mapped by exploiting the correlation between spectroscopic features and specific molecular groups. The compatibility of Fourier-transform interferometry with full-field imaging makes it the spectroscopic method of choice, but Nyquist-limited fringe sampling restricts the increments of the interferometer arm length to no more than a few microns, making the acquisition time-consuming. Here, we demonstrate a compressive hyperspectral imaging strategy that combines non-uniform sampling and a smoothness-promoting prior to acquire data at 15% of the Nyquist rate, providing a significant acquisition-rate improvement over state-of-the-art techniques. By illuminating test objects with a sequence of suitably designed light spectra, we demonstrate compressive hyperspectral imaging across the 700–1400 cm-1 region in transmission mode. A post-processing analysis of the resulting hyperspectral images shows the potential of the method for efficient non-destructive classification of different materials on painted cultural heritage.

Full-field imaging for non-invasive spectral analysis of pigments in painted cultural heritage is demonstrated from 1400–700cm-1. Acquisition without compressive sensing takes 4 minutes, with a further 2 minutes needed for spectral reconstruction and clustering.

We demonstrate compressive spectroscopy at 3.3 µm by projecting a sparse set of spectra shaped by a digital micromirror device. Acquisition at 280 Hz is achieved. Reconstructed spectroscopy shows good agreement with ground truth measurements.

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

Supercontinuum generation in bulk media is not normally observed at the nJ-level pulse energies available from high-repetition-rate femtosecond oscillators. Here, we present results demonstrating how a visible supercontinuum can be produced in bulk orientation-patterned gallium phosphide from 100-MHz 1040-nm femtosecond pulses with energies of up to 32 nJ. High-order parametric gain near 550 nm, seeded by self-phase-modulated spectral sidebands, underpins this new and simple supercontinuum process which yields an output spectrum spanning from the blue/green to the red.

Orientation-patterned gallium phosphide [1] (OPGaP) is a wide-bandgap material enabling two-photonabsorption-free pumping using 1040-nm Yb-lasers, and such OPGaP optical parametric oscillators (OPOs) have been applied in Fourier-transform [2] and dual-comb [3] spectroscopies across the 5–12-μm molecular fingerprint region. Hetero-epitaxial growth of GaP on orientation-patterned gallium arsenide (OPGaAs) templates has been developed [4] , to mitigate issues with GaP substrates, but no nonlinear frequency conversion has yet been demonstrated. Here we report the first efficient frequency conversion in OPGaP-on-OPGaAs. OPGaP layers up to 1.2-mm thick were grown by hydride vapor-phase epitaxy (HVPE) on a 3-inch MBE-grown OPGaAs template. Several multi-grating and fan-out crystals of lengths 1.1 mm and 2.9 mm were diced from the wafer and polished into plane-parallel chips, and a broadband anti-reflection coating was applied to both faces.

Spectral shaping of ultrafast laser pulses in the 3.6-3.9-pm wavelength region is implemented using a CaF2-windowed digital micromirror device. Short-pass, long-pass, bandpass and multi-wavelength shaping is demonstrated, targeting applications in spectroscopy and compressive sensing.

Unmanned aerial vehicles (UAVs)-or drones-present compelling new opportunities for airborne gas sensing in applications such as environmental monitoring, hazardous scene assessment, and facilities' inspection. Instrumenting a UAV for this purpose encounters trade-offs between sensor size, weight, power, and performance, which drives the adoption of lightweight electrochemical and photo-ionisation detectors. However, this occurs at the expense of speed, selectivity, sensitivity, accuracy, resolution, and traceability. Here, we report on the design and integration of a broadband Fourier-transform infrared spectrometer with an autonomous UAV, providing ro-vibrational spectroscopy throughout the molecular fingerprint region from 3 - 11 µm (3333 - 909 cm

Sensors are routinely mounted on robots to acquire various forms of measurements in spatio-temporal fields. Locating features within these fields and reconstruction (mapping) of the dense fields can be challenging in resource-constrained situations, such as when trying to locate the source of a gas leak from a small number of measurements. In such cases, a model of the underlying complex dynamics can be exploited to discover informative paths within the field. We use a fluid simulator as a model, to guide inference for the location of a gas leak. We perform localization via minimization of the discrepancy between observed measurements and gas concentrations predicted by the simulator. Our method is able to account for dynamically varying parameters of wind flow (e.g., direction and strength), and its effects on the observed distribution of gas. We develop algorithms for off-line inference as well as for on-line path discovery via active sensing. We demonstrate the efficiency, accuracy and versatility of our algorithm using experiments with a physical robot conducted in outdoor environments. We deploy an unmanned air vehicle (UAV) mounted with a CO2 sensor to automatically seek out a gas cylinder emitting CO2 via a nozzle. We evaluate the accuracy of our algorithm by measuring the error in the inferred location of the nozzle, based on which we show that our proposed approach is competitive with respect to state of the art baselines., Accepted as a journal paper at IEEE Robotics and Automation Letters (RA-L)

Summary The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems., Graphical Abstract, Highlights • Cascades with elongated crRNA spacers support the formation of up to 57-bp-long R-loops • Target recognition and DNA degradation require only wild-type-sized, ∼30-bp-long R-loops • Complexes with elongated crRNAs bind targets with significantly increased affinity, Songailiene et al. show that engineered versions of Cascade with elongated crRNAs can base-pair with target DNA over 57 bp. However, target recognition only requires wild-type-sized base-pairing of ∼30-bp length. These findings define constraints for improving the specificity of these complexes in biotechnological applications.

We present an eye-safe active FTIR spectroscopy system based on a broadband ultrafast optical parametric oscillator (OPO) operating in the 3.1–3.5-μm wavelength range (where common hydrocarbons display absorption peaks) and capable of acquiring gas absorption spectra from a simple target. We demonstrate that simultaneous quantitative measurement of atmospheric background levels of water and methane is readily achieved by this system, even in the presence of strong absorption from a control cell of 1.5% ethane.

Atmospheric gas sensing using unmanned aerial vehicles (UAVs) is growing in importance because of the wide range of applications this technology can flexibly address, such as climate monitoring [1], pollution detection [2], volcanology [3], and hazardous scene assessment [4]. The benefits over conventional approaches are striking: drones can be deployed quickly at low cost and without the need for dedicated runways, can operate in contaminated or hazardous environments, and can perform systematic high-resolution surveys in three-dimensional space with a high repeatability. Here we report the first intrinsically multi-species gas sensing using a UAV-integrated Fourier-transform spectrometer with long-wavelength infrared spectral coverage extending deep into the molecular fingerprint region.

We report for the first time nonlinear frequency conversion—specifically optical parametric oscillation—in OP-GaP layers grown by hydride vapor-phase epitaxy on 3-inch OP-GaAs templates. Continuous tuning with wavelength coverage from 3.9–12 µm was achieved by using stepped and fan-out gratings having periods from 18.0–35.2 µm, which propagated 150 µm of a 1.2-mm-thick layer before overgrowth. Anti-reflection-coated OP-GaP crystals were pumped at 1040 nm with an ultrafast Yb-fiber laser, yielding idler output powers up to 150 mW (60 mW) in spectra centered at a wavelength of 5.6 µm (10.7 µm).

Supercontinuum generation from nanojoule femtosecond lasers is well known in photonic-crystal fibers, channel waveguides, and micro-resonators, in which strong confinement shapes their dispersion and provides sufficient intensity for self-phase modulation, four-wave mixing, and Raman scattering to cause substantial spectral broadening. Until now, supercontinuum generation in bulk media has not been observed at equivalent energies, but here we introduce a new mechanism combining second- and third-order nonlinearities to produce broadband visible light in orientation-patterned gallium phosphide. A supercontinuum from the blue/green to the red is produced from 32 nJ 1040 nm femtosecond pulses, and a nonlinear-envelope-equation model including χ ( 2 ) and χ ( 3 ) nonlinearities implies that high-order parametric gain pumped by the second-harmonic light of the laser and seeded by self-phase-modulated sidebands is responsible.

The broad and slowly varying spectral features of liquids and solids require broadband sources in the mid- to long-wave infrared for their detection and identification. We present here a range of measurements made using uniquely tunable femtosecond optical parametric oscillators, which have enabled stand-off Fourier-transform spectroscopy to be implemented across a large part of the spectral fingerprint region. In this way we have achieved active stand-off detection of liquids on surfaces, of powders and of airborne liquid particles in aerosol form. We discuss the optical parametric oscillator technology, the spectroscopy implementations and the detection capabilities and limitations of the techniques.

Skoltech Ph.D. program in the Life Sciences (to A.K.); European Research Council consolidator grant [GA 724863 to R.S].; NIH [R01 GM10407] and Russian Science Foundation [14-14-00988] grants to K.S.; UMNIK grant 8115GU/2015 to O.M., and institutional support from Skoltech to K.S. Funding for open access charge: Skolkovo Institute of Science and Technology internal funding. In type I CRISPR-Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade-crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade-crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade-crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA. Publisher PDF

Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR-Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping.

Open-path remote sensing is critical for monitoring fugitive emissions from industrial sites, where a variety of volatile organic compounds may be released. At ranges of only a few tens of metres, spatially coherent broadband mid-infrared sources can access sufficiently large absorption cross-sections to quantify hydrocarbon gas fluctuations above ambient background levels at high signal:noise ratios. Here we report path-integrated simultaneous concentration measurements of water, methane and ethane implemented in the 3.1–3.5-µm range using 0.05-cm−1-resolution Fourier-transform spectroscopy with an ultrafast optical parametric oscillator and a simple, non-compliant target. Real-time concentration changes were observed at a range of 70 m by simulating a fugitive emission with a weak localized release of 2% methane in air. Spectral averaging yielded a methane detection sensitivity of 595 ppb·m, implying a system capability to resolve few-ppb concentrations of many volatile organic compounds at observation ranges of 50–100 m.

The rapidly developing semiconductor industry demands constant innovations in optoelectronic imaging of semiconductor integrated circuits to keep up with continuing device scaling. It was recently shown that two-photon laser-assisted device alteration (2pLADA) can deliver precision fault isolation. Here we describe an investigation into the influence of the incident light polarization on the 2pLADA spatial resolution. Linear polarization provides a highly confined but elliptical focal spot, while circular polarization diminishes the lateral resolution but benefits from a symmetrical focal spot. Radially polarized light potentially provides the highest lateral imaging resolution in all directions at the expense of the longitudinal resolution. By comparing 2pLADA results obtained using linear, circular and radial polarizations we show that certain polarizations have advantages in particular applications. Therefore a polarization optimized 2pLADA tool can achieve a sufficiently high performance to isolate faults of transistors separated by as little as 100 nm and maybe smaller.

Laser-Micro/Nanofabricated 3D Polymers for Tissue Engineering ApplicationsA multi-photon polymerization system has been designed based on a pulsed irradiation light source (diode-pumped solid state femtosecond laser Yb:KGW, 300 fs, 1030 nm, 1-200 kHz) in combination with large working area and high precision linear motor driven stages (100×100×50 mm3). The system is intended for high resolution and throughput 3D micro- and nanofabrication and enables manufacturing the polymeric templates up to 1 cm2areas with sub-micrometer resolution. These can be used for producing 3D artificial polymeric scaffolds to be applied for growing cells, specifically, in the tissue engineering. The bio-compatibility of different acrylate, hybrid organic-inorganic and biodegradable polymeric materials is evaluated experimentallyin vitro. Variously sized and shaped polymeric scaffolds of biocompatible photopolymers with intricate 3D geometry were successfully fabricated. Proliferation tests for adult rabbit myogenic stem cells have shown the applicability of artificial scaffolds in biomedicine practice.

Laser two-photon polymerization (LTPP) has been widely reported as a tool for three-dimensional micro/nanofabrication. Femtosecond lasers are employed to form nanostructures in photosensitive resins with subwavelength resolution. We demonstrate high throughput large scanning area LTPP system based on linear motor driven stages combined with Yb:KGW high repetition rate (312.5 kHz) amplified laser as irradiation source (515 nm second harmonic’s wavelength). Femtosecond green light can be focused to a smaller diffraction limited spot and provides higher structuring resolution comparing to commonly used Ti:sapphire lasers (operating at NIR wavelengths) used for LTPP. Additionally, shorter irradiation wavelength enables to process more of widely used photosensitive materials. The system capacitates production of nanostructures having 200 nm lateral resolution with high repeatability. By modifying focusing optics there is a possibility to scale up the fabrication: reduction of resolution results in shortening of fabrication time. The system enables formation of 3D structures with size varying from tens of microns to tens of millimetres. Most of the materials commonly used for photopolymerization technology (various blends of acrylates, hybrid organic–inorganic materials, and epoxy resins) are well suitable for processing with the constructed LTPP system. Keywords: two-photon absorption, laser processing, photopolymerization, micro/nanofabrication, three-dimensional structures, tissue engineering

Laser Two-Photon Polymerization (LTPP) is a technique enabling formation of 3D nanostructures in photosensitive resins with sub-wavelength resolution and unmatched flexibility. However, controllable fabrication of sub-100 nm features by this technique is still a challenge. Self-polymerization, also known as non-local polymerization, is considered to be promising in this ultra-high resolution structure formation. Recent observation of fragile self-polymerized fibres with diameter within tens of nanometres (nano-fibres) encourages the use of self-polymerization to produce nanometre scale structures other than fibres and to define the conditions for controllable fabrication. “X”-shaped polymerized supports are used as rigid structures to produce suspended self-polymerized features of different nature (shape and dimensionality) in-between the walls of “X”. By laser writing lines parallel to the substrate and perpendicular to the long symmetry axis of “X” under different conditions, selfformation of periodic nano-fibres (diameter

Femtosecond Laser Induced Polymerization is an attractive direct writing technique for rapid three-dimensional (3D) micro and nanofabrication in diverse applications. Recently, it has been successfully applied for 3D scaffold fabrication required in biomedicine applications. However, there are still a lot of investigations to be done before it can be used for practical applications in tissue engineering or regenerative medicine. In this work, experimental results on production of artificial polymeric scaffolds for stem cell growth are presented. Parameters (average laser power, sample scanning speed, and developing conditions) for microfabrication in biocompatible photopolymers AKRE (AKRE37) and ORMOSIL (SZ2080) are experimentally determined. 3D custom form and size artificial scaffolds were successfully microfabricated. Adult stem cell growth on them was investigated in order to test their biocompatibility. The results of myogenic stem cell culture expansion were compared to the control growth of the same cells on the scaffolds manufactured out of commonly used biocompatible photopolymers ORMOCER (Ormocore b59) and Poly-Ethylen Glycol Di-Acrylate (PEG-DA-258). Preliminary results show FLIP technique to have potential in fabrication of artificial 3D polymeric scaffolds for cell proliferation experiments. These are the first steps in transferring FLIP fabrication method from laboratory tests to flexible manufacturing of individual scaffolds out of biocompatible and biodegradable polymers. Keywords: femtosecond laser, two-photon polymerization, 3D micro-fabrication, biocompatible photopolymers, artificial scaffolds

CRISPR-Cas systems provide adaptive immunity against invading genetic elements such as phages and plasmids by degrading the invader DNA. In type I CRISPR-Cas systems the intrinsic RNA component of the Cascade effector complex recognizes a complementary DNA target strand (protospacer) through base pairing while displacing the non-target strand of the duplex. The resulting RNA-DNA hybrid is called R-loop. R-loop establishment recruits the helicase-nuclease Cas3, which subsequently degrades the target DNA. Here we use single-molecule DNA supercoiling experiments to investigate the recognition and verification of the target sequence by Cascade. To this end we carefully explore the effect of mutations across the target. We observe the occurrence of intermediate R-loops which lengths correspond exactly to the distance of the mutation from the start of the target sequence. These intermediate R-loops are unstable and their stability directly correlates with their length. When point mutations are eventually overcome, a full R-loop forms and becomes “locked” through conformational changes of the Cascade complex. These observations provide direct evidence for a directional R-loop formation and suggest a dynamic target scanning by Cascade, mediated through R-loop intermediates. Early encountered mutations challenge R-loop propagation and cause its collapse while distal mutations enable more stable and longer lived R-loops to overcome them. The target validation takes place exclusively at the end of the sequence when full R-loop formation induces conformational changes that initiate DNA degradation. Such a dynamic and directional R-loop propagation scheme offers several advantages for suitable homologous target recognition: (i) the complex spends little time on wrong targets that from the beginning on carry mismatches; (ii) however it supports that point mutations can be tolerated thus precluding invading DNA to escape degradation through protospacer mutations.

SummaryCRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

Nucleic acids symposium series 43(16), 8100 - 8110(2015). doi:10.1093/nar/gkv768, Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5′-W/CCGGW-3′ (W stands for A or T, ‘/’ denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an ‘open’ configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes., Published by Oxford Univ. Press, Oxford

Comparison of high-resolution sub-surface microscopy shows that illumination with linear polarization resolves an edge with resolutions of 95 nm and 120 nm, depending on E-field orientation, while radial polarization achieves a resolution of 98 nm.

Solid-immersion lens (SIL) microscopy requires the dimensions of the SIL to be precisely fabricated, otherwise spherical aberrations will be encountered [1]. Uncorrected spatial aberrations not only degrade the lateral and axial resolutions of a sub-surface microscopy system, but also couple into the time domain to stretch the pulses beyond their minimum transform-limited durations [2]. Chromatic aberration remaps the arrival times at the focal plane of the different colors in the pulse, while spherical aberration changes the position of the best focal plane for each frequency of the pulse. Therefore, time-domain measurements can be applied to diagnose the presence of spatial aberrations, and even to inform an experimental strategy of minimizing their impact. Here we describe an investigation of this approach in the context of sub-surface SIL microscopy of integrated circuits.

We investigate different linear effects that appear in the light beam propagation behind a three-dimensional woodpile photonic crystal. On one hand we report and analyse experimental observation of narrow and well collimated laser beam formation behind the photonic crystal. We show that this collimation depends on the input laser beam focusing conditions. On the other hand we also report the first theoretical and numerical observation double imaging formation behind the photonic crystal. That conclusion comes from the idea that the spatial dispersion possess multiple branches (Bloch branches), generally of different curvatures, for a fixed frequency.

We report and analyze experimental observation of the formation of a narrow, well collimated laser beam at optical frequency behind the woodpile photonic crystal fabricated using a femtosecond laser multi-photon polymerization technique. We show that the collimation depends on the input laser beam focusing conditions. We discuss the experimental results and give theoretical interpretation.

We experimentally observe formation of narrow laser beams behind the woodpile photonic crystal, when the beam remains well collimated in free propagation behind the crystal. We show that the collimation depends on the input laser beam’s focusing conditions, and we interpret theoretically the observed effect by calculating the spatial dispersion of propagation eigenmodes and by numerical simulation of paraxial propagation model.

Three-dimensional photonic crystals with woodpile structure are demonstrated to exhibit collimation of light beams behind the photonic structure. Woodpile structure photonic crystals with longitudinal modulation periods in a range of 7.4 – 7.8 µm, and transverse periods of 1 µm were fabricated using laser multi-photon polymerization technique in a negative-tone photoresist. As expected from theoretical predictions, the beams propagating along the axis of period variation collimate behind the crystal, which is a first experimental observation of the phenomenon.

A tightly focused ultrafast pulsed laser beam is guided into the volume of the photosensitive material and induces nonlinear photomodification. By translating the sample, the position of the focus is changed relatively, thus point-by-point complex 3D structures can be written inside the bulk. In this report, we present a Laser Two-Photon Polymerization (LTPP) setup for three-dimensional micro/nanostructuring for applications in photonics, microoptics, micromechanics, microfluidics and biomedicine. This system enables fabrication of functional devices over a large area (up to several cm in lateral size) with reproducible sub-micrometer resolution (up to 200 nm). In our experiments a Yb:KGW active media laser oscillator (75 fs, 200 kW, 515 nm frequency doubled, 80 MHz) was used as an irradiation source. The sample was mounted on XYZ wide range linear motor driven positioning stages having 10 nm positioning resolution. These stages enable an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support a linear scanning speed of up to 300 mm/s. Control of all the equipment was automated via custom made computer software "3D-Poli" specially designed for LTPP applications. The model of the structure can be imported as CAD file, this enables rapid and flexible structuring out of various photopolymers like ORMOCERs, ORMOSILs, acrylates and PEGDAs which are commonly used in conventional UV mask, nanoimprint and μ-stereolithographies. In this paper, we demonstrate polymeric microstructures fabricated over a large area on glass, plastic and metal substrates. This opens a way to produce functional devices like photonic crystals, microlenses, micromechanic and microfluidic components and artificial scaffolds as templates for cell growth. Additionally, results of primary myogenic stem cells expanding on microfabricated polymeric scaffolds are provided. Cell proliferation tests show the material and structure to be biocompatible for the biomedical practice.

Femtosecond laser photo-polymerization of zirconium-silicon based sol-gel photopolymer SZ2080 is used to fabricate micro-optical elements with a single and hybrid optical functions. We demonstrate photo-polymerization of the solid immersion and Fresnel lenses. Gratings can be added onto the surface of lenses. The effective refractive index of polymerized structures can be controlled via the volume fraction of polymer. We used woodpile structure with volume fraction of 0.65-0.8. Tailoring of dispersion properties of micro-optical elements by changing filling ratio of polymer are discussed. Direct write approach is used to form such structures on a cover glass and on the tip of an optical fiber. Close matching of refractive indices between the polymer and substrate in visible and near infra red spectral regions ( n SZ 2080 = 1.504, n glass = 1.52) is favorable for such integration. The surface roughness of laser-polymerized resits was ~30 nm (min-max value), which is acceptable for optical applications in the visible range. For the bulk micro-optical elements the efficiency of 3D laser polymerization is increased by a factor ~ (2 - 4) × 10 2 times (depends on the design) by the shell-formation polymerization: ( i ) contour scanning for definition of shell-surface, ( ii ) development for removal of nonfunctional resist, and ( iii ) UV exposure for the final volumetric polymerization of an enclosed volume.

Light initiated liquid polymer quasi-instant solidification is attractive for its ultra precise spatial and temporal control of the reaction. Here we present femtosecond laser induced two-photon photopolymerization for structuring of microoptical and sample photonic devices. Due to nonlinear phenomena the fabrication resolution is not restricted to diffraction limit for the applied laser excitation wavelength but determined by the exposure dose. Furthermore, pinpoint structuring enables one to produce 3D structures of any form out of photopolymer. The smallest structural elements voxels of 200 nm lateral dimensions can be achieved reproducibly by using high numerical aperture optics. Axial resolution which is fundamentally few times worse than lateral can be controlled in few hundred nanometers precision by forming polymeric pad as an additional structure. In our work we applied commercially available and widely used hybrid zirconium-silicon based hybrid sol-gel material ORMOSIL (SZ2080) and an acrylate based AKRE37 photopolymer. Arrays of custom parameters spherical microlenses for microscopy applications have been fabricated. Their surface roughness, focal distance and imaging quality were tested. 3D custom form woodpile structures with submicron period and chain-mail structure were made as a sample photonic bandgap structures. Finally, we show some structures produced out of fluorescent dyes rhodamine 6G doped photopolymer.

Two-photon polymerization has emerged as a technology for rapid fabrication of three-dimensional micro-structures with nanoscale resolution. Commonly, a Ti:Sapphire femtosecond laser (operating at 780-800 nm wavelength) working at MHz pulse repetition rate is applied as an irradiation source to photomodify the resin. We present a system for pinpoint two-photon polymerization which utilizes second harmonic (515 nm) of amplified Yb:KGW femtosecond laser working at 312.5 kHz pulse repetition rate. Shorter irradiation wavelength enables one to focus laser beam to a smaller spot. High repetition rate and high average power capacitates rapid fabrication of three-dimensional structures over a large area. Results obtained prove the highest resolution of fabrication to be up to ~100 nm, and reproducible resolution 200 nm. Some micro-structures fabricated rapidly over millimeter area and revealing the specific problems arising at high speed fabrication over large lateral dimensions are presented in this report. Results obtained show the system and technologies applied to be well suitable for future routine 3D structuring over the large area and application of such structures in photonics, micro-optics, micromechanics, microelectronics and cell growth for tissue engineering.

In this paper we demonstrate femtosecond laser fabrication of micro-tubes with a height of several tens of micrometers in the photopolymer SZ2080 by three different methods: direct laser writing, using the optical vortex beam and holographic lithography. The flexibility of direct laser writing and dramatic increase of production efficiency by applying the vortex-shaped beam and four-beam interference approaches are presented. Sample arrays of micro-tubes were successfully manufactured applying all three methods and the fabrication quality as well as efficiency of the methods is compared. The processing time of a single micro-tube with 60 ?m height and 3 ?m inner radius is reduced 400 times for the holographic lithography technique and 500 times for the optical vortex method compared with the direct laser writing technique. The processing time of a micro-tube array containing 400?micro-tubes is the shortest for the holographic lithography method but not for the optical vortex method as in the case of a single micro-tube, because the holographic lithography method does not require time for sample translation. Additionally, the holographic lithography enables manufacturing of the whole micro-tube array by a single exposure. Although point-by-point photo-structuring ensures unmatched complexity of manufactured microstructures, employing nowadays high repetition rate amplified femtosecond lasers combined with beam shaping or several beam interference can envisage industrial applications for practical demands.

Imaging Fourier transform spectroscopy (IFTS) is an established tool for spectroscopic applications due to its high-throughput, inherently broadband and high-resolution detection capability. However, applications in the long-wave infrared (LWIR) require complex cooled detection systems, increasing the cost and the scale of instrumentation. Uncooled microbolometer cameras offer a cheap and compact alternative, but at the expense of detectivity and response time. Therefore, such cameras can be implemented only in the step-scan IFTS [1] . This approach offers the unique ability to employ compressive sampling techniques to reduce acquisition rate to below the Nyquist frequency, f Nyq . In this work we combine step-scan IFTS with compressive sampling techniques to rapidly obtain hyperspectral images of plastics and art samples in the LWIR band.