Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.

Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.

The efficiency of transfer of gases and particles across the air-sea interface is controlled by several physical, biological and chemical processes in the atmosphere and water which are described here (including waves, large-and small-scale turbulence, bubbles, sea spray, rain and surface films). For a deeper understanding of relevant transport mechanisms, several models have been developed, ranging from conceptual models to numerical models. Most frequently the transfer is described by various functional dependencies of the wind speed, but more detailed descriptions need additional information. The study of gas transfer mechanisms uses a variety of experimental methods ranging from laboratory studies to carbon budgets, mass balance methods, micrometeorological techniques and thermographic techniques. Different methods resolve the transfer at different scales of time and space; this is important to take into account when comparing different results. Air-sea transfer is relevant in a wide range of applications, for example, local and regional fluxes, global models, remote sensing and computations of global inventories. The sensitivity of global models to the description of transfer velocity is limited; it is however likely that the formulations are more important when the resolution increases and other processes in models are improved. For global flux estimates using inventories or remote sensing products the accuracy of the transfer formulation as well as the accuracy of the wind field is crucial. IntroductionThe transfer of gases and particles across the air-sea interface depends not only on the concentration difference between the water and the air, but also on the efficiency of the transfer process. The efficiency of the transfer is controlled by complex interaction of a variety of processes in the air and in the water near the interface. Here we treat both gases and particles since the transfer, to some extent, is governed by similar mechanisms. Studies of transfer across the air-sea interface include a variety of methods and techniques ranging from laboratory studies, modeling and large-scale field studies. Various methods reach somewhat different conclusions, due to representation of different

[1] Thermographic techniques are presented that directly measure the temperature difference across the thermal boundary layer at the sea surface, the probability density function of surface renewal, the net heat flux, and the heat transfer velocity during nighttime. The techniques are based on a model of surface renewal. Through the use of digital image processing techniques, temporally and spatially highly resolved measurements are feasible, limited only by the thermal imager. We present laboratory measurements from the Heidelberg Aeolotron and field measurements from the GasExII cruise taken at a spatial resolution of 3 mm and temporal resolution of 10 ms. The net heat flux estimates of the thermographic techniques and micrometeorological methods agree with an error less than 5% for conditions in which the surface renewal model is applicable. Experimental evidence is presented for the probability density function of surface renewal to be best described by a logarithmic normal distribution. At moderate and high wind speeds when the influence of surface films is not significant, surface renewal seems to be an adequate model for air-water heat exchange.

Duchenne muscular dystrophy (DMD) is a common inherited muscle disease showing chronic inflammation and progressive muscle weakness. Absent dystrophin renders sarcolemma more Ca(2+) -permeable, disturbs signalling and triggers inflammation. Sustained degeneration/regeneration cycles render muscle cytoarchitecture susceptible to remodelling. Quantitative morphometry was introduced in living cells using second-harmonic generation (SHG) microscopy of myosin. As the time course of cellular remodelling is not known, we used SHG microscopy in mdx muscle fibres over a wide age range for three-dimensional (3D) rendering and detection of verniers and cosine angle sums (CASs). Wild-type (wt) and transgenic mini-dystrophin mice (MinD) were also studied. Vernier densities (VDs) declined in wt and MinD fibres until adulthood, while in mdx fibres, VDs remained significantly elevated during the life span. CAS values were close to unity in adult wt and MinD fibres, in agreement with tight regular myofibril orientation, while always smaller in mdx fibres. Using SHG 3D morphometry, we identified two types of altered ultrastructure: branched fibres and a novel, previously undetected 'chaotic' fibre type, both of which can be classified by distinct CAS and VD combinations. We present a novel model of tissue remodelling in dystrophic progression with age that involves the transition from normal to chaotic to branched fibres. Our model predicts a ~50% contribution of altered cytoarchitecture to progressive force loss with age. We also provide an improved automated image algorithm that is suitable for future ageing studies in human myopathies.

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, flow velocities and concentrations in an imaging mode. Here we extend this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular interactions in an imaging mode. We present a theoretical framework for SPIM-FCCS fitting models, which is subsequently used to evaluate several test measurements of in-vitro (labeled microspheres, several DNAs and small unilamellar vesicles) and in-vivo samples (dimeric and monomeric dual-color fluorescent proteins, as well as membrane bound proteins). Our method yields the same quantitative results as the well-established confocal FCCS, but in addition provides unmatched statistics and true imaging capabilities.

[1] Heat is used as a proxy tracer for gases to study the transport processes across the sea surface microlayer. Infrared imaging techniques permit fast measurements of heat transfer velocities and give an insight into the transport mechanisms across the thermal sublayer. The observed fluctuations of the sea surface temperature suggest that surface renewal is the major turbulent transport mechanism at medium and high wind speeds. The scale space analysis of the temperature patterns at the sea surface with respect to their contribution to the skin-bulk temperature difference shows the turbulent nature of the transport process. Large-scale turbulence dominates the transport at low friction velocities, whereas small-scale turbulence is more dominant at higher wind friction. The skin-bulk temperature difference is estimated by fitting the measured sea surface temperature distribution with a PDF function based on a surface renewal model. Periodic heat flux switching in the wind-wave flume delivers independent estimates of surface and bulk temperature and verifies the statistical approach, whereas at very low wind speeds and film-covered surfaces the statistical method underestimates the skin-bulk temperature difference across the thermal sublayer. The large scatter of the transfer velocities when plotted versus wind speed indicates that not only the wind shear but also other processes such as the wave field and surfactants influences near-surface turbulence and thus airwater gas transfer.

ABSTRACT. We investigated the large-scale (10-1000 m) and small-scale (mm-cm) variations in size, number and arrangement of air bubbles in the EPICA Dronning Maud Land (EDML) (Antarctica) ice core, down to the end of the bubble/hydrate transition (BHT) zone. On the large scale, the bubble number density shows a general correlation with the palaeo-temperature proxy, d d 18O, and the dust concentration, which means that in Holocene ice there are fewer bubbles than in glacial ice. Small-scale variations in bubble number and size were identified and compared. Above the BHT zone there exists a strong anticorrelation between bubble number density and mean bubble size. In glacial ice, layers of high number density and small bubble size are linked with layers with high impurity content, identified as cloudy bands. Therefore, we regard impurities as a controlling factor for the formation and distribution of bubbles in glacial ice. The anticorrelation inverts in the middle of the BHT zone. In the lower part of the BHT zone, bubble-free layers exist that are also associated with cloudy bands. The high contrast in bubble number density in glacial ice, induced by the impurities, indicates a much more pronounced layering in glacial firn than in modern firn.