FlavobacteriumIR1 is a gliding bacterium with a high degree of colonial organization as a 2D photonic crystal, resulting in vivid structural coloration when illuminated.Enterobacter cloacaeB12, an unrelated bacterium, was isolated from the brown macroalgaFucus vesiculosusfrom the same location as IR1. IR1 was found to be a predator of B12. A process of surrounding, infiltration, undercutting and killing of B12 supported improved growth of IR1. A combination of motility and capillarity facilitated the engulfment of B12 colonies by IR1. Predation was independent of illumination. Mutants of IR1 that formed photonic crystals less effectively than the wild type were reduced in predation. Conversely, formation of a photonic crystal was not advantageous in resisting predation byRhodococcusspp. PIR4. These observations suggest that the organization required to create structural colour has a biological function (facilitating predation) but one that is not directly related to the photonic properties of the colony. This work is the first experimental evidence supporting a role for this widespread type of cell organization in theFlavobacteriia.

Light-sheet fluorescence microscopy (LSFM) is a non-invasive optical method for the observation of living specimens. Although this concept was established a century ago it is only during the last decade that instruments suitable for biological applications have been developed, thereby circumventing some of the limitations of established fluorescence imaging techniques such as confocal laser scanning microscopy. LSFM utilizes a sheet of laser light to illuminate only a thin slice of a fluorescently labeled sample. A wide-field fluorescence microscope, placed perpendicular to the light-sheet, serves to collect the fluorescence signal and image the observed region by means of a camera. This side-on illumination configuration features several advantages, including intrinsic optical sectioning without the need of spatial filtering as employed in confocal microscopy, excellent signal-to-noise ratio, high temporal resolution, and drastically reduced overall photobleaching and phototoxicity inside living specimens. Moreover, the non-conventional geometry of LSFM opens up a completely new way of sample mounting, enabling convenient multi-view image acquisition for 3D imaging by simple rotation of the sample within the medium-filled chamber. Although LSFM was developed originally for the observation of large organisms such as zebrafish embryos, this method can be adapted to a large range of samples from macroscopic specimens like corals or copepods to microscopic organisms like tintinnids.

Fluorescence correlation spectroscopy (FCS) is a confocal microscopy-based method allowing to assess diffusion, transport and interaction properties of molecules (proteins, nucleic acids, compounds) in vitro and in vivo. Commercially available instruments enable routine measurements at one or few specific points inside living cells. FCS experiments inside living cells or embryos remain a challenge since point measurements often feature large errors caused by the heterogeneous environment of the sample. Moreover, biological noise due to cell-to-cell differences of physical and biological parameters (viscosity, protein expression) induces further variations that are difficult to separate from measurement error. Currently, these problems are addressed by performing statistical data analysis of measurements from many cells. To significantly improve the method, FCS measurements can be conducted simultaneously in many points per cell. Here we present a recently introduced microscopy setup [1] that allows spatially resolved FCS measurements in 2D optical sections across cells. The setup is based on a thin diffraction-limited light sheet that illuminates a cross-section of the cell. An EM-CCD camera placed perpendicular to the light sheet provides thousands of point detectors and enables to record in each pixel the incoming photons with single photon sensitivity, sub-millisecond time resolution and close-to-confocal spatial resolution. We have used this and a confocal setup to perform measurements of the diffusion- and binding-related mobility of chromatin-forming and -associated GFP-labeled proteins inside nuclei of living cells in interphase. We could identify and characterize the binding of heterochromatin protein 1 and of histone proteins to chromatin as well as the local dynamics of the chromatin fiber in different nuclear localisations, supporting the existence of subchromosomal domains with distinct properties. [1] Capoulade, J., Wachsmuth, M., Hufnagel, L. & Knop, M. Nat. Biotechnol. 29 (2011), 835-839.

Imaging the spatial and temporal complexity of molecules in living cells and tissues could provide important data for a quantitative understanding of biology. Capoulade et al . introduce a light sheet–based microscope that performs fluorescence correlation spectroscopy at each pixel of an image to provide spatially resolved maps of protein dynamics.

Fluorescence (cross-)correlation spectroscopy (FCS/FCCS) and generally fluorescence fluctuation spectroscopy (FFS) are confocal microscopy-based methods that allow to assess diffusion and transport properties as well as interactions of molecules (proteins, nucleic acids, compounds) in vitro and in vivo. Commercially available instrumentation enables routine measurements at one or few specific points inside living cells. However, conventional FCS/FCCS experiments remain challenging because point measurements in a living cell are associated with large error caused by the heterogeneous environment of the cellular interior. Moreover, biological noise due to cell-to-cell variations of physical and biological parameters (e.g. intracellular viscosity, protein expression levels) induces further variations, which are difficult to separate from the measurement error. Currently, these problems are partially addressed by performing statistical data analysis of measurements from many different cells. However, it is desirable to obtain more reliable and robust data from single cells with spatial resolution. This requires a new approach allowing to perform simultaneous measurements and to circumvent the problems associated with confocal FFS: photobleaching, out-of-focus illumination and loss of spatial definition due to cell movements. Here, we present a novel microscope that allows spatially resolved FFS measurements in 2D optical sections across cells. The setup is based on a single plane illumination microscope in which a thin diffraction-limited light sheet is used to illuminate a cross-section of the cell. The use of an electron-multiplying charge-coupled device (EM-CCD), placed perpendicular to the light sheet, with hundreds of single pixel detectors instead of an avalanche photodiode (a single pixel detector) enables to record on each pixel the incoming photons with single photon sensitivity and sub-millisecond time resolution. This is predicted to significantly reduce the error associated with single point measurements. It should also provide access to spatially resolved measurements of concentrations, interactions and mobilities.

The mechanism of laser induced damage in optical materials under high power nanosecond laser irradiation is commonly attributed to the presence of precursor centers. Depending on material and laser source, the precursors could have different origins. Some of them are clearly extrinsic, such as impurities or structural defects linked to the fabrication conditions.In most cases the center size ranging from sub-micrometer to nanometer scale does not permit an easy detection by optical techniques before irradiation. Most often, only a post mortem observation of optics permits to proof the local origin of breakdown.Multi-scale analyzes by changing irradiation beam size have been performed to investigate the density, size and nature of laser damage precursors. Destructive methods such as raster scan, laser damage probability plot and morphology studies permit to deduce the precursor densities. Another experimental way to get information on nature of precursors is to use non destructive methods such as photoluminescence and absorption measurements.The destructive and non destructive multiscale studies are also motivated for practical reasons. Indeed LIDT studies of large optics as those used in LMJ or NIF projects are commonly performed on small samples and with table top lasers whose characteristics change from one to another. In these conditions, it is necessary to know exactly the influence of the different experimental parameters and overall the spot size effect on the final data.In this paper, we present recent developments in multiscale characterization and results obtained on optical coatings (surface case) and KDP crystal (bulk case).

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

We describe in this paper a model to link laser damage initiators properties (nature, size distribution, density) to measured Laser Induced Damage Threshold (LIDT). It is based oil calculation of light absorption in nanoabsorbers and subsequent heating, coupled to laser damage statistics in order to obtain the laser damage probability as a function of laser fluence. Applications to the case of optical coatings are then presented. We study the influence of laser irradiation parameters and coatings properties oil LIDT measurements. By coupling this multiscale study to our model, we show that information oil the initiating defects properties and the physical damage mechanisms involved call be obtained: discrimination between different defects, estimation of densities, size and nature of defects, evolution of the defect's under multiple irradiation. Implication of this approach for physical understanding and metrology applications are discussed.

A comparative study is made on the laser damage resistance of monolayers coatings made with different technologies. HfO2 and SiO2 thin films have been deposited on fused silica substrates with Dual Ion Beam Sputtering, Electron Beam Deposition (with and without Ion Assistance) and Reactive Low Voltage Ion Plating technologies. The laser damage thresholds of these coatings have been determined at 1064nm and 355nm using a nanosecond pulsed YAG laser, and a 1-on-1 test procedure.

Chalcogenide coatings are investigated to obtain either optical components for spectral applications or optochemical sensors in the mid-infrared. The deposition of Ge(15)Sb(20)S(65) and Te(20)As(30)Se(50) chalcogenide glasses is performed by two physical techniques: electron-beam and pulsed-laser deposition. The quality of the film is analyzed by scanning electron microscopy, atomic force microscopy, and energy dispersive spectroscopy to characterize the morphology, topography, and chemical composition. The optical properties and optical constants are also determined. A CF(4) dry etching is performed on these films to obtain a channeled optical waveguide. For a passband filter made by electron-beam deposition, cryolite as a low-refractive-index material and chalcogenide glasses as high-refractive-index materials are used to favor a large refractive-index contrast. A shift of a centered wavelength of a photosensitive passband filter is controlled by illumination time.

We have investigated the influence of laser beam size on laser-induced damage threshold (LIDT) in the case of single- and multiple-shot irradiation. The study was performed on hafnia thin films deposited with various technologies (evaporation, sputtering, with or without ion assistance). LIDT measurements were carried out at 1064 nm and 12 ns with a spot size ranging from a few tens to a few hundreds of micrometers, in 1-on-1 and R-on-1 modes. These measurements were compared with simulations obtained with the statistical theory of laser-induced damage caused by initiating inclusions. We show how to obtain information on the initiating defect properties and the related physical damage mechanisms with a multiscale study. Under certain conditions, it is possible with this method to discriminate different defects, estimate their densities, and follow the evolution of the defects under multiple irradiation. The different metrology implications of our approach, particularly for obtaining a functional LIDT of optical components are discussed.

A major issue in the use of high-power lasers, such as the Laser Megajoule (LMJ), is laser-induced damage of optical components. One potential damage initiator is particulate contamination, but its effect is hard to distinguish from that of other damage precursors. To do so, we introduced artificial contaminants typical of metallic pollution likely to be present on the optical components of the LMJ chains. More precisely, aluminum particles of two different sizes were placed on a silica sample. These dots were characterized by optical microscopy and profilometry. Then they were exposed to a laser beam with a pulse length of 6.5 ns at 1064 nm and fluences in the range from 1 to 40 J/cm(2). Each dot was characterized again with the same techniques and also by photothermal microscopy. To complete the experimental results, we performed numerical simulations with a one-dimensional Lagrangian hydrodynamics code. We show that the particle removal by laser irradiation produces a modification of the silica surface that does not evolve into catastrophic damage under subsequent irradiation. However, the effect does depend on the size of the dots. We demonstrate that a procedure exists that removes the dot and leaves the site capable of resisting high fluence.

The laser resistance of large optical components remains an important limitation for the performances and the maintenance costs in LMJ or NIF projects. For practical reasons LIDT studies are commonly performed with small samples and table top lasers whose characteristics change from one to another. In these conditions, it is necessary to know exactly the influence of the different experimental parameters (wavelength, spot size,....) on the final data. These considerations are particularly true in heterogeneous materials as KDP crystals. Indeed the use of different laser beam sizes (from mu m to hundreds mu m) to plot laser damage probability curves had clearly shown that at 355nm in KDP, it is possible to exhibit a limit of irradiated area which permit to distinguish two different LIDT associated with two laser damage precursors densities. This prior result has put in evidence the influence of irradiated beam size in the discrimination of different kinds of defects in KDP.We present in this paper a systematic study of beam size effect in KDP for three different wavelengths: 355nm, 532nm and 1064nm. This study performed in 1: 1 and R: I mode will reveal precursors for each wavelength and their respective evolution under repetitive shots for small and large beams. This multi-parameters study will help us to highlight mechanisms involved in laser-induced damage in KDP crystal.

We propose a model to link laser damage initiator properties (such as nature, size distribution, and density) to measured laser damage probabilities in optical materials. The model is based on the calculation of light absorption in nanoabsorbers and subsequent heating, coupled to laser damage statistics, and allows to obtain the laser damage probability as a function of laser fluence. Applications to the case of optical coatings irradiated in the nanosecond regime are presented. Laser damage probability curves are measured in hafnia single layer coatings made under different conditions: electron beam deposition and reactive low voltage ion plating. By studying the influence of the laser irradiation parameters (wavelength and beam size) and coating properties on the simulations, we show with our methodology that initiating defects (hafnium inclusions) can be identified. The implications of this approach for physical understanding and metrology applications are discussed.

For large aperture solid state lasers, the laser resistance of the optical component remains an important limitation for the performances and the maintenance costs. Since decades, laser induced damage has been intensively studied in order to understand and control the origin of the phenomenon. LID measurements are commonly performed with table top lasers whose characteristics change from one to another and, sometimes, the scaling laws do not permit to explain the experimental differences. For example, we have previously demonstrated that, in KH2PO4 (KDP) crystals, the laser beam size can influence strongly the determination of the damage probability. Here, we present a systematic study realized on KDP crystal to quantify the influence of the beam size on the LIDT (Laser Induced Damage Threshold) measurement at 355 nm. The use of an unique Gaussian beam ranged from micronic to sub-millimetric sizes permits to highlight different types of laser-damage precursor. LIDT measurements realized with beams of small (lower than 100 pm at 1/e(2))or large (upper than 400 mu m at 1/e2)dimensions give information about the behavior of material regarding precursor defects.

The damage mechanisms in silica thin films exposed to high fluence 1064nm nano-second laser pulses are investigated. The thin films under study are made with different techniques (evaporation and sputtering, with and without ion assistance) and the results are compared. The material morphological, optical and structural modifications are locally analyzed with optical microscopy and profilometry, photoluminescence and absorption microscopies. These observations are made for fluences near and above the laser damage threshold, and also in the case of multiple pulse irradiations. An increase in absorption in and around the damages is observed, as well as the generation of different defects that we spatially resolve with absorption and luminescence mappings.

The influence of the laser beam size on the laser-induced damage threshold (LIDT) in thin films and substrates is investigated. LIDT measurements realized with beam of different dimensions give information on laser damage precursors.