We present a method for imaging the optical near-fields of nanostructures, which is based on the local ablation of a smooth silicon substrate by means of a single, femtosecond laser pulse. At those locations, where the field enhancement due to a nanostructure is large, substrate material is removed. The resulting topography, imaged by scanning electron or atomic force microscopy, thus reflects the intensity distribution caused by the nanostructure at the substrate surface. With this method one avoids a possible distortion of the field distribution due to the presence of a probe tip, and reaches a resolution of a few nanometers. Several examples for the optical near-field patterns of dielectric and metallic nanostructures are given. The optical properties of nanostructures are an important issue in nanoscience with a tremendous potential for applications. This holds both for individual particles and particle arrays, and for dielectric materials (e.g., photonic crystals) as well as metals (e.g., optical antennas).1-3 The optical nearfields of such particles, which are essential for understanding their function, are not easily accessible by experimental means. One approach is to use a scanning near-field optical microscope to image the intensity distribution in the vicinity of the nanostructures with a fine aperture.4 Different approaches have improved the resolution to below 25 nm. 5,6We introduce here an alternative method that consists in imaging optical near-field intensities by means of intense short laser pulses. This technique also reaches a resolution of a few nanometers, much smaller than the laser wavelength used, but which, moreover, is not hampered by possible distortions of the resulting patterns due to the presence of a probe tip.In our experiments the nanoparticles, located on a smooth substrate, are irradiated with a single, femtosecond laser pulse, which is perpendicularly incident onto the substrate plane. The intensity of the pulse is adjusted to a value sufficiently low that the parts of the substrate far away from the particles are not affected. Nevertheless, the substrate surface under and near a particle will be ablated if the local intensity enhancement in the optical near-field is high enough.7 Thus, the resulting ablation pattern in the substrate, which can be imaged by scanning electron or atomic force microscopy (AFM), represents a nonlinear "photograph" of the optical near-field intensity distribution of the nanoparticle under study. Because of the short duration of the laser pulse, a smearing out of the resulting structures due to thermal conduction, as it can appear for nanosecond pulses, does not occur.Our investigations cover optical near-fields of both dielectric and metallic nanostructures. In the case of dielectric nanoparticles, we have used polystyrene spheres, available as monodisperse colloidal suspensions, with different diameters in the range of a few hundred nanometers. These particles were deposited on a silicon substrate (commercial silicon wafer) by means of spin coatin...

The butterfly genus Hypolimnas features iridescent blue colouration in some areas of its dorsal wings. Here, we analyse the mechanisms responsible for such colouration on the dorsal wings of Hypolimnas salmacis and experimentally demonstrate that the lower thin lamina in the white cover scales causes the blue iridescence. This outcome contradicts other studies reporting that the radiant blue in Hypolimnas butterflies is caused by complex ridge-lamellar architectures in the upper lamina of the cover scales. Our comprehensive optical study supported by numerical calculation however shows that scale stacking primarily induces the observed colour appearance of Hypolimnas salmacis.

For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC-sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high-throughput ultrastructural imaging of substantial volumes.Lay DescriptionTo look at immune cells from zebrafish we employed array tomography, a technique where arrays of serial sections deposited on solid substrates are used for imaging. Cell populations were isolated from the different organs of zebrafish involved in haematopoiesis, the production of blood cells. They were chemically fixed and centrifuged to concentrate them in a pellet that was then dehydrated and embedded in resin. Using a custom-built handling device it was possible to place hundreds of serial sections on silicon wafers as well ordered arrays. To image a whole cell at a resolution that would allow identifying all the organelles (i.e. compartments surrounded by membranes) inside the cell, stacks of usually 50–100 images were recorded in a scanning electron microscope (SEM). This recording was either done manually or automatically using the newly released Atlas Array Tomography platform on a ZEISS SEM. For the imaging of the sections a pixel size of about 5 nm was chosen, which defines membrane boundaries very well and allows segmentation of the membrane topology. After alignment of the images, cellular components were segmented to locate the individual organelles within the 3D reconstruction of the whole cell and also to create an inventory of organelles. Based on their morphologies we could identify specific cell types in the different hematopoietic organs. We could also quantify the proportion of each cell type in the whole population isolated from a given organ.Some of these specific cells from zebrafish were grown in a culture dish together with human cancer cells. By time-lapse light microscopy we observed that the fish cells attacked the cancer cells and killed them. From this we concluded that these cells must be similar to the cytotoxic cells from humans that play an important role in defence against spontaneously arising cancer cells in our bodies. They form special structures, called immunological synapses that we could also identify on our arrays and reconstruct in 3D. This is the first time the potential of zebrafish immune cells to form immunological synapses has been demonstrated.Our study is a good example for the practicality ...

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