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A comprehensive understanding of the mechanisms leading to chronic inflammation of tissues after exposure to different types of nanomaterials is greatly lacking. In the case of lung tissue, repeating events of exposure to metal-oxide or carbon nanomaterials can eventually lead to persistent inflammation and further cardiovascular diseases [1,2]. A similar outcome can occur in periprosthetic tissues suffering severe inflammation from the constant influx of metal wear debris from nearby implant materials [3]. To better understand these adverse outcomes, one needs to dig into the initial events that are formed on a molecular, nanoscale, thus requiring an advanced combination of microscopy techniques. Lately, more and more studies in live science are tackled by correlative microscopy (CM) which implements an optimal combination of complementary and advanced techniques on the same sample to be able to reveal new phenomena. In our recent studies related to both lung and periprosthetic tissue inflammation, we show the new workflow of advanced microscopies and spectroscopies from which we have gained new structural as well as functional insights (Figure 1) [4,5]. With the combination of super-resolution optical microscopy (STED), hyperspectral and fluorescence lifetime imaging (sp-FLIM), Helium Ion Microscopy (HIM), Scanning electron microscopy (SEM-EDS), and Proton Induced X-ray Emission (PIXE) we reveal/present the new mechanisms and impact of nanomaterial interaction with lung epithelium and periprosthetic tissue, which leads to better knowledge and causal relations of the nanotoxicity on such small scales. References: 1. X. Li, L. Jin, H. Kan, Nature 2019, 570, 437-439. 2. E. Underwood, Science 2017, 355, 342–345. 3. S.B. Goodman, J. Gallo, E. Gibon, M. Takagi, Expert Rev Med Devices 2020, 17, 41–56. 4. H. Kokot, et. al, Advanced Materials, 2020, 32, 2003913-1-15. 5. R. Podlipec, et. al, Materials, 2021, 14, 3048.

Knowing the temperature distribution within the conducting walls of various multilayer-type materials is crucial for a better understanding of heat-transfer processes. This applies to many engineering fields, good examples being photovoltaics and microelectronics. In this work, we present a novel fluorescence technique that makes possible the non-invasive imaging of local temperature distributions within a transparent, temperature-sensitive, co-doped Er:GPF1Yb0.5Er glass-ceramic with micrometer spatial resolution. The thermal imaging was performed with a high-resolution, fluorescence microscopy system, measuring different focal planes along the z-axis. This ultimately enabled a precise axial reconstruction of the temperature distribution across a 500-µm-thick glass-ceramic sample. The experimental measurements showed excellent agreement with computer-modeled heat simulations and suggest that the technique could be adopted for the spatial analyses of local thermal processes within optically transparent materials. For instance, the technique could be used to measure the temperature distribution of intermediate, transparent layers of novel ultra-high-efficiency solar cells at the micron and sub-micron levels.

Comprehensive understanding of molecular events governing lung epithelium response to inhaled nanoparticles is still lacking. These repeating events in lungs could eventually lead to persistent inflammation and further cardiovascular diseases [1,2]. To better understand how it all start on a molecular, nanoscale level one urgently needs an appropriate model system and an advanced imaging technique(s) capable of unravelling key information on a nanoscale. Many studies addressing such complex biological problems have been lately tackled by correlative microscopy (CM) approach using an optimal combination of complementary and advanced techniques [3]. Our approach was thus to apply live cell epithelium model imaging using an advanced high-resolution fluorescence microscopy followed by helium ion microscopy (HIM) to visualize structures and morphology further down at nano-scale. One of the HIM advantages to other high-resolution, high-vacuum imaging techniques is large depth of focus, sub-nm resolution, nm surface sensitivity, and especially no need for sample coating that changes the nanostructure morphology on the surface. To gather any further structure-function information of the investigated biological system using such diametrical techniques, an appropriate sample preparation needed to be developed. Once done, we could study sub-micron to nanometer changes that govern model lung epithelium interaction with various nanoparticles. Exposure of metal oxide (TiO2) nanotubes has revealed active passivation of nanomaterial-biological matter composites on the cell surface with lipo-proteins present, identified both with optical and ion beam technique (Figure, below). Findings of CM studies have contributed to better understand chronic inflammation prediction in lung diseases [4]. On the other hand, exposed carbon nanoparticles have shown completely different cell response and will be discussed. 1. Li, X., Jin, L. & Kan, H. Air pollution: a global problem needs local fixes.

Helium Ion Microscopy (HIM) has not been thoroughly exploited for biological studies addressing relevant questions that range from the cellular to the subcellular level. One of the benefits of HIM compared to other high-resolution imaging techniques is definitely the large depth of focus, sub-nm resolution, nm surface sensitivity, and especially that no sample coating is needed that can change the nanostructure morphology on the surface. The prerequisite to getting the most from the technique is thus the appropriate sample preparation. Besides, to get the most from understanding the addressed biological question, a successful correlative microscopy approach is necessary. This is best shown in our recently published study (H Kokot, Advanced Materials, 2020), where we have addressed one of the most critical issues in toxicology, that is the poor understanding of chronic inflammation initiation in lung tissue caused by inhaled nanoparticles, with the correlative microscopy approach using advanced multimodal optical microscopy and HIM. HIM nicely revealed the TiO2 nanotube organization and passivation on the cell surface and confirmed lipid and protein binding to the TiO2 surface (Figure below), identified as well by in silico simulations. In brief, HIM is an extremely powerful technique for the surface, and in the case of porous samples also in-depth morphology characterization on an nm scale. In combination with complementary imaging techniques and proper sample preparation, many relevant biological questions can be addressed and solved. Still, there are many limitations and challenges in cell preparation and imaging using helium ions, such as imaging of internal structures, definitely pursuing discussions and new developments in the future.

Clear understanding of molecular events followed by lung epithelial cells/tissue response to inhaled nanoparticles is still lacking. As these interaction events in lungs eventually lead to diseases and potentially persistent inflammation [1,2], one urgently needs new and relevant investigation methods which could provide new insights into the key mechanisms of interaction. In our latest research we have thus focused on this toxicology problem first by developing an appropriate in vitro lung epithelial model and second by developing and implementing relevant advanced correlative imaging techniques capable of gathering more insight of interaction properties on scales well below optical resolution limit. In order to understand the mechanisms of molecular initiative events we have first performed live cell imaging using STED super-resolution microscopy by which few tens to hundred nm resolution was achieved locally. As the technique is incapable of providing resolution further down to nm and lacks the visualization of non-labeled surrounding structures and morphology, we thus introduced suitable complementary correlative microscopy techniques with high surface contrast, SEM and Helium Ion Microscopy (HIM). Main focus, besides sample and sample holder preparation for these high vacuum techniques, was dedicated to HIM measurements which in general are capable of providing better resolution and sensitivity compared to SEM [3]. From this ongoing study we briefly present the first interesting results of correlative microscopy combining optical, electron and ion based techniques on the epithelial cells exposed to TiO2 nanoparticles from micro to nano scale. References: 1. Li, X., Jin, L. & Kan, H. Air pollution: a global problem needs local fixes. Nature 570, 437–439 (2019). 2. Underwood, E. The polluted brain. Science 355, 342–345 (2017). 3. Hlawacek, G. et Al. Helium Ion Microscopy. J. Vac. Sci. Technol. 32, (2014)

Clear understanding of molecular events followed by lung epithelial cells/tissue response to inhaled nanoparticles is still lacking. As these interaction events in lungs eventually lead to diseases and potentially persistent inflammation [1,2], one urgently needs new and relevant investigation methods which could provide new insights into the key mechanisms of interaction. In our latest research we have thus focused on this toxicology problem first by developing an appropriate in vitro lung epithelial model and second by developing and implementing relevant advanced correlative imaging techniques capable of gathering more insight of interaction properties on scales well below optical resolution limit. In order to understand the mechanisms of molecular initiative events we have first performed live cell imaging using STED super-resolution microscopy by which few tens to hundred nm resolution was achieved locally. As the technique is incapable of providing resolution further down to nm and lacks the visualization of non-labeled surrounding structures and morphology, we thus introduced suitable complementary correlative microscopy techniques with high surface contrast, SEM and Helium Ion Microscopy (HIM). Main focus, besides sample and sample holder preparation for these high vacuum techniques, was dedicated to HIM measurements which in general are capable of providing better resolution and sensitivity compared to SEM [3]. From this ongoing study we briefly present the first interesting results of correlative microscopy combining optical, electron and ion based techniques on the epithelial cells exposed to TiO2 nanoparticles from micro to nano scale. References: 1. Li, X., Jin, L. & Kan, H. Air pollution: a global problem needs local fixes. Nature 570, 437–439 (2019). 2. Underwood, E. The polluted brain. Science 355, 342–345 (2017). 3. Hlawacek, G. et Al. Helium Ion Microscopy. J. Vac. Sci. Technol. 32, (2014)

Correlative microscopy combining light and electron microscopy (CLEM) has become one of the important and unmissable tools in various investigations of complex biological systems revealing high-resolution structural and highly-specific functional information [1]. In the last years more combinations of other advanced techniques have been developed, such as combining optical microscopy with atomic force spectroscopy/microscopy (AFM) or with magnetic resonance imaging, etc [2] whereas in our study multimodal optical microscopy has been correlated with ion and electron based techniques such as is helium ion microscopy (HIM) [3]. The purpose for using combination of the complementary techniques was to elucidate or better interpret specific biological problems which could not be explained just by one technique lacking of whether resolution, sensitivity of specificity. We focused on toxicology related scientific questions of how inhaled nanoparticles interact with lung epithelial cells/tissue once get into direct contact and why interactions can eventually lead to diseases and potentially persistent inflammation [4,5]. In order to better understand the interaction on nanometer scales we first developed proper lung in-vitro model which was followed by proper sample preparation for efficient correlative microscopy using multimodal optical microscopy and high resolution HIM microscopy. By latter we managed to image single metal oxide nanoparticles on cell surfaces interacting with cell membranes, while functional information of the same events was prior measured with confocal and super-resolution optical microscopy. Besides, we implemented described correlative microscopies also for scientific problem related to rejection of hip implants where material debris is found everywhere in the surrounding periprosthetic tissue with lack of knowledge what happens on a molecular scale. The latest findings of both ongoing studies will be presented. References: 1. P. de Boer, JP Hoogenbo

Photocatalytic degradation of dichloroacetic acid (DCA) was studied in a continuous-flow set-up using a titanium microreactor with an immobilized double-layered TiO2 nanoparticle/nanotube film. Chloride ions, formed during the degradation process, negatively affect the photocatalytic efficiency and at a certain concentration (approximately 0.5 mM) completely stop the reaction in the microreactor. Two proposed mechanisms of inhibition with chloride ions, competitive adsorption and photogenerated-hole scavenging, have been proposed and investigated by adsorption isotherms and electron paramagnetic resonance (EPR) measurements. The results show that chloride ions block the DCA adsorption sites on the titania surface and reduce the amount of adsorbed DCA molecules. The scavenging effect of chloride ions during photocatalysis through the formation of chlorine radicals was not detected.

Site-directed spin labeling (SDSL) electron spin resonance (ESR) spectroscopy is a relatively new biophysical tool for obtaining structural information about proteins. This thesis presents a novel approach, based on powerful spectral analysis techniques (multicomponent spectral simulations and evolutionary optimizations of ESR spectra) and modeling of the protein structure by calculating the restrictions of the conformational space of the attached spin label. First, the feasibility of the ESR spectral analysis was enhanced by speeding-up the spectrum optimization and by automation of the analysis routines to enable the handling of large sets of spectroscopic data (e.g., for the joint analysis of SDSL-ESR spectra from multiple sites of a spin-labeled protein). According to the testing examples a speed-up factor of 5-7 was achieved. Secondly, SDSL-ESR was used to study the topology of the long N-terminal domain of the photosynthetic light-harvesting complex CP29. Wild-type protein containing a single cysteine at position 108 and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases the apoproteins were either solubilized in detergent, or they were reconstituted with their native pigments in vitro. The spin label ESR spectra were analyzed in terms of a multi-component spectral simulation approach. These results permit to trace the structural organization of the long Nterminal domain of CP29 leading to a structural model for its N-terminal domain. Thirdly, we proposed a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. The proposed structural model takes into account the restricting effect of the protein backbone, amino acid side chains an

The topology of the long N-terminal domain (100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain

Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch.