Christoph Pratsch

The choice of optical labels for live-cell imaging requires a characterization of interactions with their biological environment. Here, we discuss alterations of the molecular composition and interactions in the endolysosomal pathway of living cells by the fluorescent dye for lysosome labeling LysoSensor Green (LSG) based on vibrational nanospectroscopy using surface-enhanced Raman scattering (SERS) and soft X-ray nanotomography (SXT). A connection between the accumulation of LSG and ultrastructural changes is found by SXT that suggests an impact of the dye on cellular function. The biochemical effects in the endolysosomal system are detailed by different SERS experiments using gold nanoparticles as plasmonic probes, one that includes endolysosomes of all maturation stages and another one probing exclusively the lysosomal stage as the target compartment of the fluorescent label. The differences in SERS signals between the cells exposed to LSG compared to their controls are related to changes in the biochemical environment of the gold nanoparticles in the endolysosomal structures. As indicated by the occurrence of specific signals assigned to the interaction of the nanoprobes with proteins, nucleic acids, lipids, and LSG, respectively, the presence of LSG mainly affects the activity of enzymes that are involved in the degradation of proteins and nucleic acids in the lysosomes. The molecular changes when endolysosomes of all maturation stages contribute to the spectrum further point to LSG affecting cellular function along the whole endolysosomal pathway. The proposed complementary approach for nanoprobe characterization can be used for the validation of existing optical labels in fluorescence and multimodal imaging methods.

Ceramide is a central bioactive lipid that acts as both a membrane structural component and a crucial signaling molecule in the cells. In order to maintain cellular homeostasis, ceramide metabolism is tightly regulated by enzymes in the endolysosomal system such as acid ceramidase (AC) and acid sphingomyelinase (ASM). We investigate the biochemical consequence of ceramide accumulation within the endolysosomes of living animal cells by optical nanoprobing, using surface-enhanced Raman scattering (SERS) with gold nanoparticles. The ceramide level in 3T3 fibroblast cells was systematically increased by interfering with two key enzymatic pathways in sphingolipid metabolism as well as by adding exogenous ceramide. The modulation of enzyme activity occurred by the inhibition of AC using the inhibitor N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]2-chloroacetamide (SACLAC) in different incubation schemes and supplementation of the cells with additional ASM, respectively, both were added through the culture medium. The analysis of SERS data from the endolysosomal compartment reveals changes in the structure and interaction of proteins alongside variations in membrane composition and organization that correspond to ceramide stress. Combined cryo soft-X-ray nanotomography data of the intact cells show that the biomolecular alterations transform the cellular ultrastructure to varying degrees depending on the specific route and extent of ceramide increase. The ultrastructural changes include severe membrane deformation and changed vesicular organization as a consequence of a high ceramide content. The results demonstrate the label-free optical monitoring of metabolic processes at the subcellular level, before their complex biochemical background, and refine the description of molecular and nanostructure changes associated with distorted sphingolipid metabolism.

Ceramide synthase (CerS) is a key enzyme in sphingolipid metabolism, predominantly maintaining lipid homeostasis within the endoplasmic reticulum (ER). Capturing the biochemical consequences of CerS activity at the subcellular level is of great interest to understand complex lipid flux in living cells. Here, surface-enhanced Raman scattering (SERS) combined with soft X-ray tomography and random forest (RF) machine learning was employed to probe the cellular response to CerS inhibition. The nanospectroscopy data from endolysosomes and changes in ultrastructure demonstrate diverging alterations upon the sequential administration of CerS inhibitor Fumonisin B1 and SERS probe gold nanoparticles. RF analysis reveals distinct spectral patterns of the affected endolysosomes, characteristic of a ceramide imbalance under different conditions, indicating a tight metabolic coupling between the ER and endolysosomes. The vibrational nanospectroscopy coupled with computational reconstruction of molecular interaction patterns and ultrastructural imaging provides a comprehensive strategy to understand enzyme activity and metabolic dysregulation in situ.

In electron microscopy, obtaining low-noise images is often difficult, especially when examining biological samples or delicate materials. Therefore, the suppression of noise is essential for the analysis of such noisy images. State-of-the-art image denoising methods are dominated by supervised Convolution neural network (CNN)-based methods. However, supervised CNNs cannot be used if a noise-free ground truth is unavailable. To address this problem, we propose a method that uses re-occurring patterns in images. Our proposed method does not require noise-free images for the denoising task. Instead, it is based on the idea that averaging images with the same signal having independent noise suppresses the overall noise. In order to evaluate the performance of our method, we compare our results with other state-of-the-art denoising methods that do not require a noise-free image. We show that our method is the best for retaining fine image structures. Additionally, we develop a confidence map for evaluating the denoising quality of the proposed method. Furthermore, we analyze the time complexity of the algorithm to ensure scalability and optimize the algorithm to improve the runtime efficiency.

Understanding a catalytic reaction requires tools that elucidate the structure of the catalyst surface and subsurface, ideally at atomic resolution and under reaction conditions. Operando electron microscopy meets this requirement in some cases, but fails in others where the required reaction conditions cannot be reached or lead to an unwanted influence of the electron beam on the reactant and catalyst. We introduce ILIAS (identical location imaging and spectroscopy) in combination with a quasi in situ approach to disentangle the effect of heat and gas on the surface of nanoparticles from the effect of the electron beam. With this approach we allow high temperatures and pressures in any gaseous environment on the one hand, and atomic resolution imaging and spectroscopy on the other. As a proof of concept, we resolve the structural evolution of a Co₃O₄ spinel catalyst using ILIAS and track the oxidation state across the surface before and after heating in a reductive or oxidative environment. We then titrate the surface of the catalyst using CO as a probe molecule to remove highly active oxygen species formed during the thermal treatment, providing unprecedented insight into the interplay between pretreatment and surface reactivity of Co₃O₄ nanoparticles.

Memristive devices with valence change mechanism (VCM) show promise for neuromorphic data processing, although emulation of synaptic behavior with analog weight updates remains a challenge. Standard filamentary and area‐dependent resistive switching exhibit characteristic differences in the transition from the high to low resistance state, which is either abrupt with inherently high variability or gradual and allows quasi‐analog operation. In this study, the two switching modes are clearly correlated to differences in the microstructure and electronic structure for Pt/Al₂O₃/TiO_x/Cr/Pt devices made from amorphous layers of 1.2 nm Al₂O₃ and 7 nm TiO_x by atomic layer deposition. For the filamentary mode, operando spectromicroscopy experiments identify a localized region of ≈50 nm in diameter of reduced titania surrounded by crystalline rutile‐like TiO₂, highlighting the importance of Joule heating for this mode. In contrast, both oxide layers remain in their amorphous state for the interfacial mode, which proves that device temperature during switching stays below 670 K, which is the TiO₂ crystallization temperature. The analysis of the electronic conduction behavior confirms that the interfacial switching occurs by modulating the effective tunnel barrier width due to accumulation and depletion of oxygen vacancies at the Al₂O₃/TiO_x interface. The results are transferable to other bilayer stacks.

Co-abietate and Cu-abietate complexes were obtained by a low-cost and eco-friendly route. The synthesis process used Pinus elliottii resin and an aqueous solution of CuSO₄/CoSO₄ at a mild temperature (80 °C) without organic solvents. The obtained complexes are functional pigments for commercial architectural paints with antipathogenic activity. The pigments were characterized by Fourier-transform infrared spectroscopy (FTIR), mass spectrometry (MS), thermogravimetry (TG), near-edge X-ray absorption fine structure (NEXAFS), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and colorimetric analysis. In addition, the antibacterial efficiency was evaluated using the minimum inhibitory concentration (MIC) test, and the antiviral tests followed an adaptation of the ISO 21702:2019 guideline. Finally, virus inactivation was measured using the RT-PCR protocol using 10% (w/w) of abietate complex in commercial white paint. The Co-abietate and Cu-abietate showed inactivation of >4 log against SARS-CoV-2 and a MIC value of 4.50 µg·mL⁻¹ against both bacteria Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The results suggest that the obtained Co-abietate and Cu-abietate complexes could be applied as pigments in architectural paints for healthcare centers, homes, and public places.

Holography is a powerful method for achieving 3D images of objects. Extending this method to short wavelengths potentially offers significantly higher resolution than visible light holography. However, current X-ray holography setups employ nanoscale pinholes to form the reference wave. This approach is relatively inefficient and limited to very small sample size. Here, we propose a new setup for X-ray holography based on a binary diffractive optical element (DOE), which forms at the same time the object illumination and the reference wave. This optic is located separately from the sample plane, which permits investigation of larger sample areas. Using an extended test sample, we demonstrate a resolution of 90 nm (half-pitch) at an undulator beamline at BESSY II. The new holography setup can be directly transferred to free electron laser sources enabling time-resolved nanoscale imaging for ultra-fast processes.

The transmission X-ray microscope’s 3D PSF exhibits an axial asymmetry that cannot be explained by an incoherent imaging model for either a lens or zone plate, with or without spherical aberration.