Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

Müller M, Mönkemöller V, Hennig S, Hübner W, Huser T. Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ. Nature Communications. 2016;7(1): 10980

Survival rate of eukaryotic cells following electrophoretic nanoinjection

Simonis M, Hübner W, Wilking A, Huser T, Hennig S. Survival rate of eukaryotic cells following electrophoretic nanoinjection. Scientific Reports. 2017;7(1): 41277.Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells

Patellar Tendon Shear Wave Velocity Is Higher and has Different Regional Patterns in Elite Competitive Alpine Skiers than in Healthy Controls

Competitive alpine skiers are exposed to enormous forces acting on their bodies-particularly on the knee joint and hence the patellar tendon - during both the off-season preparation and in-season competition phases. However, factors influencing patellar tendon adaptation and regional pattern differences between alpine skiers and healthy controls are not yet fully understood, but are essential for deriving effective screening approaches and preventative countermeasures. Thirty elite competitive alpine skiers, all members of the Swiss Alpine Ski Team, and 38 healthy age-matched controls were recruited. A set of two-dimensional shear wave elastography measurements of the PT was acquired and projected into three-dimensional space yielding a volumetric representation of the shear wave velocity profile of the patellar tendon. Multivariate linear models served to quantify differences between the two cohorts and effects of other confounding variables with respect to regional shear wave velocity. A significant (p < 0.001) intergroup difference was found between skiers (mean ± SD = 10.4 ± 1.32 m/s) and controls (mean ± SD = 8.9 ± 1.59 m/s). A significant sex difference was found within skiers (p = 0.024), but no such difference was found in the control group (p = 0.842). Regional SWV pattern alterations between skiers and controls were found for the distal region when compared to the mid-portion (p = 0.023). Competitive alpine skiers exhibit higher SWV in all PT regions than healthy controls, potentially caused by long-term adaptations to heavy tendon loading. The presence of sex-specific differences in PT SWV in skiers but not in controls indicates that sex effects have load-dependent dimensions. Alterations in regional SWV patterns between skiers and controls suggest that patellar tendon adaptation is region specific. In addition to the implementation of 3D SWE, deeper insights into long-term tendon adaptation and normative values for the purpose of preventative screening are provided

MoNa - a cost-efficient, portable system for the nanoinjection of living cells

Simonis M, Sandmeyer A, Greiner J, Kaltschmidt B, Huser T, Hennig S. MoNa - a cost-efficient, portable system for the nanoinjection of living cells. Scientific reports. 2019;9(1): 5480.Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells' health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC's) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes

The serotonergic central nervous system of the Drosophila larva: anatomy and behavioral function.

The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naïve odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed

Entropy-based Super-resolution Imaging(ESI): From Disorder to Fine Detail

Yahiatene I, Hennig S, Müller M, Huser T. Entropy-based Super-resolution Imaging(ESI): From Disorder to Fine Detail. ACS Photonics. 2015;2(8):1049-1056

Nanoparticles as Nonfluorescent Analogues of Fluorophores for Optical Nanoscopy

Hennig S, Mönkemöller V, Böger C, Müller M, Huser T. Nanoparticles as Nonfluorescent Analogues of Fluorophores for Optical Nanoscopy. ACS Nano. 2015;9(6):6196-6205.Optical microscopy modalities that achieve spatial resolution beyond the resolution limit have opened up new opportunities in the biomedical sciences to reveal the structure and kinetics of biological processes on the nanoscale. These methods are, however, mostly restricted to fluorescence as Contrast mechanism, which limits the ultimate spatial resolution and observation time that can be achieved by photobleaching of the fluorescent probes. Here, we demonstrate that Raman scattering provides a valuable contrast mechanism for optical nanoscopy in the form of super-resolution to structured illumination microscopy. We find that nanotags, i.e., gold and silver nanoparticles that are capable of surface-enhanced Raman scattering (SERS), can be imaged with a spatial resolution beyond the diffraction limit in four dimensions alongside and with similar excitation power as fluorescent probes. The highly polarized nature of super-resolution structured illumination microscopy renders these nanotags elliptical in the reconstructed super-resolved images, which enables us to determine their orientation within the sample. The robustness of nanotags against photobleaching allows us to image these particles for unlimited periods of time. We demonstrate this by imaging isolated nanotags in a dense layer of fluorophores, as well as on the surface of and after internalization by osteosarcoma cells, always in the presence of fluorescent probes. Our results show that SERS nanotags have the potential to become highly multiplexed and chemically sensitive optical probes for optical nanoscopy that can replace fluorophores in applications where fluorescence photobleaching is prohibitive for following the evolution of biological processes for extended times

Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns

Hennig S, van de Linde S, Bergmann S, Huser T, Sauer M. Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns. ACS Nano. 2015;9(8):8122-8130

Entropy-Based Super-Resolution Imaging (ESI): From Disorder to Fine Detail

We introduce a novel and universal method for fast optical high, as well as super-resolution imaging. Our method is based on reconstructing super-resolved images from conventional image sequences containing rapid random signal fluctuations. Such sequences could be obtained from either wide-field single-molecule blinking experiments or rapid image sequences with fluorophores undergoing random intensity fluctuations. By calculating the local entropy (<i>H</i>) and cross-entropy (x<i>H</i>) values pixel-by-pixel, weighted with higher order statistics (HOS), a new image with pixel intensities representing the true information content in the time series is obtained. We show that analyzing image sequences by this formalism enables the reconstruction of super-resolved images, where the optical resolution that can be achieved depends only on the number of input frames and the higher order moments used for the calculation. We find that the acquisition of <100 frames per sequence is sufficient to reconstruct super-resolved images of entire cells. We also demonstrate that not only on–off switching of the fluorescent dyes, but also other dynamic events, that is, photobleaching, can be exploited for efficient and high-resolution image reconstructions. This method opens up the potential to obtain super-resolved images from most wide-field fluorescence microscopy systems. By providing a universal Fiji-plugin most users of high-end fluorescence microscopy systems will now benefit from this easy-to-use super-resolution optical microscopy method