Siegfried Steltenkamp

Practical aspects of Boersch phase contrast electron microscopy of biological specimens

Implementation of physical phase plates into transmission electron microscopes to achieve in-focus contrast for ice-embedded biological specimens poses several technological challenges. During the last decade several phase plates designs have been introduced and tested for electron cryo-microscopy (cryoEM), including thin film (Zernike) phase plates and electrostatic devices. Boersch phase plates (BPPs) are electrostatic einzel lenses shifting the phase of the unscattered beam by an arbitrary angle. Adjusting the phase shift to 90° achieves the maximum contrast transfer for phase objects such as biomolecules. Recently, we reported the implementation of a BPP into a dedicated phase contrast aberration-corrected electron microscope (PACEM) and demonstrated its use to generate in-focus contrast of frozen-hydrated specimens. However, a number of obstacles need to be overcome before BPPs can be used routinely, mostly related to the phase plate devices themselves. CryoEM with a physical phase plate is affected by electrostatic charging, obliteration of low spatial frequencies, and mechanical drift. Furthermore, BPPs introduce single sideband contrast (SSB), due to the obstruction of Friedel mates in the diffraction pattern. In this study we address the technical obstacles in detail and show how they may be overcome. We use X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) to identify contaminants responsible for electrostatic charging, which occurs with most phase plates. We demonstrate that obstruction of low-resolution features is significantly reduced by lowering the acceleration voltage of the microscope. Finally, we present computational approaches to correct BPP images for SSB contrast and to compensate for mechanical drift of the BPP.

μ-biomimetic flow-sensors—introducing light-guiding PDMS structures into MEMS

In the area of biomimetics, engineers use inspiration from natural systems to develop technical devices, such as sensors. One example is the lateral line system of fish. It is a mechanoreceptive system consisting of up to several thousand individual sensors called neuromasts, which enable fish to sense prey, predators, or conspecifics. So far, the small size and high sensitivity of the lateral line is unmatched by man-made sensor devices. Here, we describe an artificial lateral line system based on an optical detection principle. We developed artificial canal neuromasts using MEMS technology including thick film techniques. In this work, we describe the MEMS fabrication and characterize a sensor prototype. Our sensor consists of a silicon chip, a housing, and an electronic circuit. We demonstrate the functionality of our μ-biomimetic flow sensor by analyzing its response to constant water flow and flow fluctuations. Furthermore, we discuss the sensor robustness and sensitivity of our sensor and its suitability for industrial and medical applications. In sum, our sensor can be used for many tasks, e.g. for monitoring fluid flow in medical applications, for detecting leakages in tap water systems or for air and gas flow measurements. Finally, our flow sensor can even be used to improve current knowledge about the functional significance of the fish lateral line.

In situ generation of electrochemical gradients across pore-spanning membranes

Silicon substrates with cavities in the micrometre range were micro-fabricated and appropriately functionalized to allow for the generation of pore-spanning membranes (PSMs) sealing the pore cavities. PSMs were either formed by applying lipids dissolved in organic solvent (painting technique) on a hydrophobically functionalized silicon surface followed by ‘solvent freeze-out’, or solvent-free PSMs were prepared by spreading of giant unilamellar vesicles on hydrophilically functionalized substrates. The geometry of the silicon cavities in conjunction with three dimensional confocal laser scanning microscopy images enabled us to simultaneously monitor the PSMs and a pH-sensitive dye entrapped into the picolitre-sized cavities. The excellent sealing properties of both PSM types allowed an in situ generation of proton gradients across these membranes. In the presence of nigericin, a proton/potassium-antiporter, a preformed potassium ion gradient was transformed into a stable proton gradient across the PSMs, which was visualized by the pH-sensitive dye pyranine entrapped in the silicon cavities in a time resolved manner by means of confocal laser scanning fluorescence microscopy.

Micro-machined flow sensors mimicking lateral line canal neuromasts

Fish sense water motions with their lateral line. The lateral line is a sensory system that contains up to several thousand mechanoreceptors, called neuromasts. Neuromasts occur freestanding on the skin and in subepidermal canals. We developed arrays of flow sensors based on lateral line canal neuromasts using a biomimetic approach. Each flow sensor was equipped with a PDMS (polydimethylsiloxane) lamella integrated into a canal system by means of thick- and thin-film technology. Our artificial lateral line system can estimate bulk flow velocity from the spatio-temporal propagation of flow fluctuations. Based on the modular sensor design, we were able to detect flow rates in an industrial application of tap water flow metering. Our sensory system withstood water pressures of up to six bar. We used finite element modeling to study the fluid flow inside the canal system and how this flow depends on canal dimensions. In a second set of experiments, we separated the flow sensors from the main stream by means of a flexible membrane. Nevertheless, these biomimetic neuromasts were still able to sense flow fluctuations. Fluid separation is a prerequisite for flow measurements in medical and pharmaceutical applications.

X-ray scattering experiments with high-flux X-ray source coupled rapid mixing microchannel device and their potential for high-flux neutron scattering investigations.

Small-angle X-ray scattering provides global, shape-sensitive structural information about macromolecules in solution. Its extension to time dimension in the form of time-resolved SAXS investigations and combination with other time-resolved biophysical methods contributes immensely to the study of protein dynamics. TR-SAXS can also provide unique information about the global structures of transient intermediates during protein dynamics. An experimental set-up with low protein consumption is essential for an extensive use of TR-SAXS experiments on protein dynamics. In this direction, a newly developed 20-microchannel microfluidic continuous-flow mixer was combined with SAXS. With this set-up, we demonstrate ubiquitin unfolding dynamics after rapid mixing with the chaotropic agent Guanidinium-HCl within milliseconds using only ∼ 40 nanoliters of the protein sample per scattering image. It is suggested that, in the future, this new TR-SAXS platform will help to increase the use of time-resolved small-angle X-ray scattering, wide-angle X-ray scattering and neutron scattering experiments for studying protein dynamics in the early millisecond regime. The potential research field for this set-up includes protein folding, protein misfolding, aggregation in amyloidogenic diseases, function of intrinsically disordered proteins and various protein-ligand interactions.Graphical abstract

Towards an optimum design for electrostatic phase plates

Charging of physical phase plates is a problem that has prevented their routine use in transmission electron microscopy of weak-phase objects. In theory, electrostatic phase plates are superior to thin-film phase plates since they do not attenuate the scattered electron beam and allow freely adjustable phase shifts. Electrostatic phase plates consist of multiple layers of conductive and insulating materials, and are thus more prone to charging than thin-film phase plates, which typically consist of only one single layer of amorphous material. We have addressed the origins of charging of Boersch phase plates and show how it can be reduced. In particular, we have performed simulations and experiments to analyze the influence of the insulating Si3N4 layers and surface charges on electrostatic charging. To optimize the performance of electrostatic phase plates, it would be desirable to fabricate electrostatic phase plates, which (i) impart a homogeneous phase shift to the unscattered electrons, (ii) have a low cut-on frequency, (iii) expose as little material to the intense unscattered beam as possible, and (iv) can be additionally polished by a focused ion-beam instrument to eliminate carbon contamination accumulated during exposure to the unscattered electron beam (Walter et al., 2012, Ultramicroscopy, 116, 62-72). We propose a new type of electrostatic phase plate that meets the above requirements and would be superior to a Boersch phase plate. It consists of three free-standing coaxial rods converging in the center of an aperture (3-fold coaxial phase plate). Simulations and preliminary experiments with modified Boersch phase plates indicate that the fabrication of a 3-fold coaxial phase plate is feasible.

Novel method for a flexible double-sided microelectrode fabrication process

Flexible devices with integrated micro electrodes are widely used for neuronal as well as myogenic stimulation and recording applications. One main intention by using micro electrodes is the ability of placing an appropriate amount of electrodes on the active sites. With an increasing number of single electrodes the selectivity for signal acquirement and analysis is significantly improved. The further advantage of small and elastic structures inside the biological tissue is the perfect fit. This lead to lower traumatisation of the nerve and muscle fibres during and after acute and chronically surgery. Different designed and structured flexible micro electrodes have been developed at the IBMT based on polyimide as substrate material over the last years including cuff, intrafascicular and shaft electrodes. All these systems are generally built up as single sided devices which reduce the possible electrode site half the area. Especially for shaft and intrafascicular applications having double sided electrode arrangement would increase the selectivity enormous. So areas on both sides can be monitored simultaneously. Recent developments of double sided flexible electrode systems lead to promising results especially for varied signal recording. Though these developments revealed some challenges in the field of micromachining including low yield rates. In this work we describe a new technical approach to develop double sided flexible micro electrode systems with a reproducible high yield rate. Prototypes of intrafascicular and intramuscular electrode systems have been developed and investigated by the means of electrochemical characterisation and mechanical behaviour. Additional investigations have been performed with scanning electron microscopy. We also give an outlook to future in vitro and in vivo experiments to investigate the application performance of the developed systems

Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m−1, and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells.

An uncooled capacitive sensor for IR detection

The beetle Melanophila acuminata detects forest fires from distances as far as 80 miles away. To accomplish this, the beetle uses highly specific IR receptors with a diameter of approximately 15 μm. These receptors are mechanoreceptors that detect deformations induced by the absorption of radiation. Although the detection mechanism is understood in principle, it is still unclear how the beetle reaches such high sensitivity. In this work, we present the biomimetic approach of an uncooled IR sensor based on the beetle’s receptors. This sensor is based on a fluid-filled pressure cell and operates at room temperature. Upon absorbing IR radiation, the fluid heats up and expands. The expanding fluid deflects one electrode of a plate capacitor. By measuring the change in capacitance, the volume increase and the absorbed energy can be inferred. To prevent the risk of damage at high energy absorption, a compensation mechanism is presented in this work. The mechanism prevents large but slow volume changes inside the pressure cell by a microfluidic connection of the pressure cell with a compensation chamber. The channel and the compensation chamber act as a microfluidic low-pass filter and do not affect the overall sensitivity above an appropriate cut-off frequency. Using MEMS technology, we are able to incorporate the complete system into a silicon chip with an area of a few mm2. Here, we show a proof-of-concept and first measurements of the sensor.

A model for mu-biomimetic thermal infrared sensors based on the infrared receptors of Melanophila acuminata

Beetles of the genus Melanophila acuminata detect forest fires from distances as far as 130 km with infrared-sensing organs. Inspired by this extremely sensitive biological device, we are developing an IR sensor that operates at ambient temperature using MEMS technology. The sensor consists of two liquid-filled chambers that are connected by a micro-fluidic system. Absorption of IR radiation by one of these chambers leads to heating and expansion of a liquid. The increasing pressure deflects a membrane covered by one electrode of a plate capacitor. The micro-fluidic system and the second chamber represent a fluidic low-pass filter, preventing slow, but large pressure changes. However, the strong frequency dependence of the filter demands a precise characterization of its properties. Here, we present a theoretical model that describes the frequency-dependent response of the sensor based on material properties and geometrical dimensions. Our model is divided into four distinct parts that address different aspects of the sensor. The model describes the frequency-dependent behaviour of the fluidic filter and a thermal low-pass filter as well as saturation effects at low frequencies. This model allows the calculation of optimal design parameters, and thereby provides the foundation for the development of such a sensor.