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

Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

Mönkemöller V, Øie C, Hübner W, Huser T, McCourt P. Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations. Scientific Reports. 2015;5(1): 16279.Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations

Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction

Markwirth A, Lachetta M, Mönkemöller V, et al. Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction. Nature Communications. 2019;10(1): 4315.Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics

Primary rat LSECs preserve their characteristic phenotype after cryopreservation

Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver non-parenchymal cells have gained increasing attention for their potential role in the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target

Primary rat LSECs preserve their characteristic phenotype after cryopreservation

Mönkemöller V, Mao H, Hübner W, et al. Primary rat LSECs preserve their characteristic phenotype after cryopreservation. Scientific Reports. 2018;8(1): 14657.Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver nonparenchymal cells have gained increasing attention for their potential role in the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target

Optical super-resolution microscopy of the structure and dynamics of cellular nanopores

Mönkemöller V. Optical super-resolution microscopy of the structure and dynamics of cellular nanopores. Bielefeld: Universität Bielefeld; 2016

Comment on "Magnetic-field-enabled resolution enhancement in super-resolution imaging'' by M. Zhang et al., Phys. Chem. Chem. Phys., 2015, 17, 6722-6727

Bergmann S, Mönkemöller V, Huser T. Comment on "Magnetic-field-enabled resolution enhancement in super-resolution imaging'' by M. Zhang et al., Phys. Chem. Chem. Phys., 2015, 17, 6722-6727. PHYSICAL CHEMISTRY CHEMICAL PHYSICS. 2017;19(6):4887-4890.Certain fluorophores, in particular those that can undergo photoinduced radical pair reactions are known to exhibit a magnetic field dependent fluorescence summarized in the term magnetic field effect (MFE). We tried to reproduce experiments that reported magnetic field enhanced fluorescence for commonly used organic dyes with a high quantum yield suitable for single molecule localization microscopy. We find that the enhanced fluorescence is due to fluorescence reflected by the magnet's surface rather than MFE

Surface modification of LPCVD ZNO-Films for silicon thin film solar cells

Lükermann F, Mönkemöller V, Kurz H, et al. Surface modification of LPCVD ZNO-Films for silicon thin film solar cells. In: Proceedings of the 24th EU PVSEC. 2009: 2299-2303

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

Characterization of silicon thin film solar cells by electroluminescence imaging

Mönkemöller V, Gondorf A, Niederkrüger M, Heinzmann U, Stiebig H. Characterization of silicon thin film solar cells by electroluminescence imaging. LOT-Oriel; 2012