Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.Lay DescriptionThe composition of our world and our bodies on the very small scale has always fascinated people, making them search for ways to make this visible to the human eye. Where light microscopes reach their resolution limit at a certain magnification, electron microscopes can go beyond. But their capability of visualizing extremely small features comes at the cost of a very small field of view. Some of the questions researchers seek to answer today deal with the ultrafine structure of brains, bones or computer chips. Capturing these objects with electron microscopes takes a lot of time – maybe even exceeding the time span of a human being – or new tools that do the job much faster. A new type of scanning electron microscope scans with 61 electron beams in parallel, acquiring 61 adjacent images of the sample at the same time a conventional scanning electron microscope captures one of these images. In principle, the multibeam scanning electron microscope’s field of view is 61 times larger and therefore coverage of the sample surface can be accomplished in less time. This enables researchers to think about large-scale projects, for example in the rather new field of connectomics. A very good introduction to imaging a brain at nanometre resolution can be found within course material from Harvard University on http://www.mcb80x.org/# as featured media entitled ‘connectomics’.

Major progress has been achieved in recent years in three-dimensional microscopy techniques. This applies to the life sciences in general, but specifically the neuroscientific field has been a main driver for developments regarding volume imaging. In particular, scanning electron microscopy offers new insights into the organization of cells and tissues by volume imaging methods, such as serial section array tomography, serial block-face imaging or focused ion beam tomography. However, most of these techniques are restricted to relatively small tissue volumes due to the limited acquisition throughput of most standard imaging techniques. Recently, a novel multi-beam scanning electron microscope technology optimized to the imaging of large sample areas has been developed. Complemented by the commercialization of automated sample preparation robots, the mapping of larger, cubic millimeter range tissue volumes at high-resolution is now within reach. This Mini Review will provide a brief overview of the various approaches to electron microscopic volume imaging, with an emphasis on serial section array tomography and multi-beam scanning electron microscopic imaging.

Abstract. Angle-dependent dip coating (ADDC) is a modified dip coating technique that offers advantages for the production of optical interference filters. In contrast to conventional dip coating (DC), the substrate is withdrawn from the coating solution under an angle of inclination. Thereby, the two surfaces of the substrate are coated with individual film thicknesses. An experimental setup for ADDC has been built and the decisive process influences on coating thicknesses have been evaluated. In order to gain full control over the individual layer thicknesses, reaching from 20 nm to 160 nm, it is necessary to vary the following process parameters: lifting speed, angle of inclination and concentration of the dipping solution. The results of coating experiments prove the advantages of ADDC over DC. A first example aims at reducing the number of coating steps: an ADDC long pass filter produced in 10 coating steps reaches the same optical performance as a conventional DC filter made in 16 steps. A second example demonstrates the possibility to improve quality: a commercial DC beam splitter can be improved with respect to the flatness of transmission and reflection curves when being produced in 4 steps by ADDC instead of 8 steps by DC. Furthermore, ADDC offers the possibility to fabricate even narrow band pass filters, which are naturally difficult to obtain by conventional DC.

Charting and understanding the full wiring diagram of complex neuronal structures such as the central nervous system or the brain (Connectomics) is one of the big remaining challenges in life sciences. Although at first it appears nearly impossible to map out a full diagram of, e.g., a mouse brain with sufficient resolution to identify each and every connection between neurons, recent technological advances move such an ambitious undertaking into the realms of possibility without spending decades at a microscope. However there are still many challenges to address in order to pave the way for fast and systematic neurobiological understanding of whole networks. These challenges range from a more robust and reproducible sample preparation to automated image data acquisition, more efficient data storage strategies and powerful data analysis tools. Here we will review novel imaging techniques developed for the challenge of mapping out the full connectome of a nervous system, brain or eye to name just a few examples. The imaging techniques reviewed cover light sheet illumination methods, single and multi-beam scanning electron microscopy, and we will briefly mention the possible combination of both light and electron microscopy. In particular we will review 'clearing' and in vivo methods that can be performed with light sheet fluroescence microscopes such as the ZEISS Lightsheet Z.1. We will then focus on scanning electron microscopy with single and multi-beam instruments including methods such as serial blockface imaging and array tomography methods.

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