Microscopy Conference 2023

Prof.

Session chairs

Prof. Dr.

Correlative microscopy aims at a combination of different imaging modalities to analyze a single specimen by more than one microscopic technique. Recent developments in microscopy as well as in computational approaches make it more and more feasible to combine different imaging modalities in a correlative and multimodal way. Therefore, this session will focus not only on the ‘classic combination’ of light and electron microscopy but will cover also other approaches that include X-ray, Raman- or atomic force microscopy (AFM). It is also aimed to report on new computational approaches, such as machine learning to analyze the obtained CLEM data.

Intravital correlative microscopy of metastatic cancer cells at the blood-brain-barrier

LS 5: Correlative and multimodal microscopy

LS 3: Imaging of large volumes and plastic section tomography

Abstract text (incl. figure legends and references) Introduction Patients suffering from malignant melanoma, breast cancer and lung cancer are at high risk of developing brain metastases (BM), which are at this time incurable and have devasting consequences for quality of life, already at the time of the diagnosis. The underlying mechanisms of brain colonisation of the metastatic cancer cells, immune cell infiltration and interaction with the unique brain microenvironment are yet to be uncovered and are challenging to study in preclinical models. Objectives The focus of our work is to better understand how metastatic cancer cells derived from extracranial tumors surpass the blood-brain-barrier, seed the brain and how the cross-talk with immune-cells and brain-resident cells influences BM growth and development. Here, we set out to target relevant cellular processes with brain-penetrant therapeutic agents at an early stage of brain colonisation in order to prevent the formation of large, uncurable brain metastases. Materials &amp; methods Our preclinical models of breast cancer and melanoma brain metastases enable to monitor long-term the dynamic development of BM in mice using intravital microscopy (IVM) through chronic cranial window. In order to achieve high-resolution insight into these processes, we perform intravital correlative microscopy, combining IVM, x-ray micro-CT and 3D electron microscopy. Results Here, I will disuss different projects aiming to reduce BM growth in vivo by by targeting early and critical events during brain colonisation. Intravital correlative microscopy was used to reveal how metastatic cancer cells cross the blood-brain-barrier. We found that this process is MMP9 dependent, and that the main source of the MMP9 is the cancer cell itself (Karreman et al, in revision). Moreover, we demonstrate that low-dose PI3K/mTOR inhibition in a preventative setting hinders melanoma brain metastatic growth in vivo (Tehranian et al, Neuro-oncology, 2021). Finally, using newly developed mouse models, we identified a new route for T cells to enter the brain and target melanoma brain metastases (Messmer et al, submitted for publication). Conclusion There is a pressing need to develop new therapeutic approaches against BM development. Targeting the earliest steps of this disease enables to circumvent more aggressive and often ineffective treatments. Intravital (correlative) microscopy on various models of brain metastases enabled us to uncover important mechanisms of brain colonisation and thus inform new treatment strategies.

Invited talk

Uncovering new roles of actin cytoskeleton using correlative light and electron microscopy

Abstract text (incl. figure legends and references) Sterols are key lipids for biological membranes, thereby their abundance and distribution must be tightly regulated. At subcellular level, organelles such as endosomes are central hubs for sterol distribution, mediating sterol transfer between the diverse subcellular destinations. Sterols reach and leave endosomes via small vesicular carriers or via sterol transfer proteins at short-range membrane contact sites. How endosomal sterol transfer is orchestrated is poorly understood. Using a genetically encoded sterol biosensor (D4H) in the fission yeast Schizosaccharomyces pombe, we have found that sterol steady-state distribution can be re-routed from the plasma membrane to endosomal structures upon pharmacological inhibition of branched actin assembly. In yeast, branched actin plays major roles in plasma membrane reshaping, whereas in complex eukaryotes branched actin also facilitates membrane trafficking in endosomes, however the role of actin at endosomes remains unclear. We show that upon inhibition of Arp2/3 complex, essential for actin branching, endosomes undergo ultrastructural remodeling from elongated to round organelles and become sterol rich. Upon reactivation of Arp2/3 complex, sterols rapidly redistribute to the plasma membrane and endosomes regain their elongated ultrastructure. Using correlative light and electron tomography (CLEM) we have found that under Arp2/3 inhibition, endosomes are smooth ellipsoidal organelles with no evidence of active membrane remodeling. Processing cells for CLEM immediately after Arp2/3 complex re-activation revealed that endosomes undergo intense ultrastructural remodeling into morphologically complex organelles with tubulations, fenestrations and budding events. Altogether our findings suggest a novel role for branched actin facilitating membrane remodeling at early endosomes. We investigate the mode of activation of Arp2/3 at endosomes and the role of endosomal ultrastructure on sterol trafficking. We propose that branched actin is necessary for endosome tubulation, facilitating membrane remodeling by trafficking complexes and thus enabling sterol trafficking through the endosomal system.

Correlative microscopy and imaging secondary ion mass spectrometry for high resolution nano-analytics on biological tissue

IM 1: Progress in instrumentation and ultrafast EM

Abstract text (incl. figure legends and references) Introduction Applications featuring sub-20 nm-imaging and chemical analysis at the same time typically rely on electron microscopy, which can be directly coupled to energy dispersive x-ray microanalysis (EDX) or used in combination with other tools like imaging mass spectrometry. Our group develops integrated focused ion beam (FIB)-based instruments featuring electron and/or ion primary beams, coupled to a double focusing magnetic sector secondary ion mass spectrometry (SIMS) system. This allows for correlative investigation of ultrastructure and chemical mapping within the same instrument at a lateral resolution of below 15 nm1. Objectives Presentation of a selection of application fields of FIB-SIMS in biology, nanotoxicology and beyond. Material and Methods Correlative electron (SE, BSE) and SIMS imaging of biological samples was performed using integrated FIB-SIMS instruments developed at LIST (e.g. Zeiss Orion Nanofab-SIMS, FEI Scios-SIMS). They yielded highly resolved chemical maps of elemental ions, their isotopes and respective small or large cluster molecules. Localisation and identification of pristine nanoparticles, but also metals embedded in complex matrices like tissue sections are performed. Additional detectors for e.g. transmitted helium beam detection on a Gas Field Ion Source (GFIS) equipped prototype instrument, called npSCOPE2, allow also correlative TEM-like imaging and analysis capabilities with helium beam at 20 - 30 keV and at temperatures down to -145°C thanks to the incorporation of cryo-setup capabilities. Results Uptake of foodborne nanoparticles by epithelial cells or bacteria, and nanoparticular redistribution of tattoo ink in skin phantoms and internal ion distributions of physiological elements are demonstrated. Conclusion High-resolution SIMS (&lt; 15 nm resolution) enables detection of nanosized metal particles in biological tissue samples. In comparison to SEM-EDX at 10 keV, SIMS with 20-30 keV primary ion beams features both higher lateral resolution and sensitivity and provides isotopic information over a wide mass range. A cryo-workflow for the npSCOPE is currently under development (see abstract by Taubitz et al.) and will facilitate close to native chemical analysis of diagnostic, environmental and nanotoxicology samples. Funding information: This project has received funding from the European Union&quot;s Horizon 2020 Research and Innovation Programme under grant agreement No. 720964 and by the Luxembourg National Research Fund via the project INTER/DFG/19/13992454. Ink samples investigated as part of the COST COMULIS round robin study for novel correlative multimodal analyses (CA17121). References: 1 Audinot et al. Highest resolution chemical imaging based on secondary ion mass spectrometry performed on the helium ion microscope. Rep Prog Phys 2021;84(10). doi: 10.1088/1361-6633/ac1e32. 2 De Castro, O. et al. npSCOPE: A New Multimodal Instrument for In Situ Correlative Analysis of Nanoparticles. Anal.

Abstract talk

Identification and characterization of uncultivated archaeal virocells using correlative fluorescence and scanning electron microscopy

LS 7: Advances in sample preparation

Abstract text (incl. figure legends and references) Microbes and their interactions with viruses play important roles in biogeochemical cycles and shape Earth&quot;s biomes. However, information on viral interactions has largely been extrapolated from a very small fraction of cultivated exemplars – the large majority of microbes remains uncultivated. Viral infections of key environmental microbes with their respective viruses forming so-called virocells, are hard to visualize and characterize in environmental samples1. Emerging methods in fluorescence microscopy enlighten smaller and less frequent structures within microbial cells. For example, it is now possible to design fluorescent probes to target viruses infecting uncultivated hosts and identify different infection stages in environmental samples using virusFISH2. Here we introduce the correlation of virusFISH with scanning electron microscopy for locating virocells and studying their morphology in environmental samples. VirusFISH for identifying viral infections of Candidatus Altiarchaeum hamiconexum within environmental biofilms was performed as described previously2, but modified specifically to carry out the correlation. After localizing virocells using fluorescence microscopy on a gridded coverslip, samples were processed for scanning electron microscopy using osmium tetroxide and an acetone dehydration series, followed by critical point drying and coating using Pt/Pd. The gridded coverslip facilitated the correlation of the micrographs using GIMP, showing that the biofilms stayed attached to the grid throughout sample preparation. Similar to cultivation-based studies, it was possible to identify morphological changes of virocells. However unlike other archaea, where a significant increase of cell size over several orders of magnitude was observed3, Candidatus Altiarchaeum hamiconexum only shows a slight, but significant increase of cell size by 10-20%. Within virocells a spatial separation of host DNA and viral reproduction was observed allowing conclusions regarding spatial and functional organization within virocells. Besides the study of virocells itself, this study also supports previous hypotheses that cell lysis due to viral infection fuels the growth of heterotrophic organisms in the deep subsurface. We conclude that the correlation of virusFISH with electron microscopy enables the characterization of virus-host interactions in environmental samples. In the future, this approach will support hypothesizes based on in silico studies to shed like on a board range of interactions of uncultivated microorganisms. DeLong, J. P. et al. Towards an integrative view of virus phenotypes. Nat Rev Microbiol 20, 83–94 (2022). Turzynski, V., Monsees, I., Moraru, C. &amp; Probst, A. J. Imaging Techniques for Detecting Prokaryotic Viruses in Environmental Samples. Viruses 13, 2126 (2021). Liu, J. et al. Virus-induced cell gigantism and asymmetric cell division in archaea. Proceedings of the National Academy of Sciences 118, e2022578118 (2021).

Correlative and volumetric EM imaging of inflammatory dynamics in skin vessels

Abstract text (incl. figure legends and references) Introduction: Serial block-face scanning electron microscopy (SBF-SEM) increases reliability, availability and throughput of 3D-EM. The ability to resolve nanoscales with a large field of view and great depths, can provide insight to many biological fields. However, analysis of cellular dynamics remains exclusive to fluorescent markers, visualized in light microscopy. Pairing both processes in a complicated, correlative (CLEM) approach is therefore necessary and new reliable workflows have to be established, taking into consideration mismatch of resolution and targeting on nano-scale level. For instance, tissue homeostasis and its balance upon inflammatory events often lead to severe changes regarding permeability of blood vessels and transmigration of neutrophils and platelets. Neutrophils may leave the vessel in a controlled process1, whereas platelets seal the endothelial barrier, preventing vascular leakage2. Interaction dynamics of both have been observed at sites of inflammation3 but to this day lacked ultrastructural investigation. Objectives: Observe the interaction of platelets and leukocytes following an inflammation stimulus via intravital microscopy (IVM) before fixation. Relocate that specific vessel and region of interest led by artificial and natural landmarks to allow subsequent ultrastructural investigation. Adapt the fixation and staining protocol for complex, multi-layered skin samples and enable consistent SBF-SEM imaging. M&amp;M: A dorsal skinfold chamber frame was attached to anesthetized and depilated LysMeGFP mice, injected with immunolabelled PECAM-1 (vascular endothelium) and GPIbβ (platelets) and subjected to immune-complex mediated vasculitis. Neutrophil recruitment and inflammatory bleeding events within a designated stamp-labelled area were imaged for 90-120 min. Subsequent sample fixation and introduction of additional landmarks included perforation of the skin, allowing penetration of fluids, biopsy-punching the area of interest and glueing it on carbon-gridded coverslips. To enhance contrast it was crucial to increase the temperature to 40 °C during all incubation steps of the adapted protocol for SBF-SEM imaging. Beneath extensive Osmium staining, infiltration was performed with a mixture of Ketjenblack to account for charge accumulation during SBF-SEM imaging. Results: The use of natural and artificial landmarks enabled precise relocation of distinct vessels and targeted trimming. IVM in response to inflammation stimuli reveals that neutrophils leave vessels through partial openings of endothelial junctions. Additionally, rare occasions of co-interaction in which an increased number of platelets follow the transmigration of neutrophils can be observed. Adaptations to the staining protocol enables volumetric serial block-face imaging to detect complete platelets transferring the basement membrane barrier. Conclusion: Many CLEM projects remain challenging throughout all steps, reaching from sample preparation towards relocation and final imaging. The workflow presented had to be optimized step-by-step. Finally, SBF-SEM offers the ability to visualize complex, volumetric cell interactions. Combination of state-of-the-art LM tools can help guide and address relevant biological questions. Fig.1: Representative IVM MIP and SBF-SEM volume merge of neutrophil recruitment. 1Vestweber D (2015) How leukocytes cross the vascular endothelium 2Braun LJ et al. (2020) Platelets docking to VWF prevent leaks during leukocyte extravasation by stimulating Tie-2 3Lisman T (2018) Platelet-neutrophil interactions as drivers of inflammatory and thrombotic disease

LS 5
Correlative and multimodal microscopy

Appointment

Session chairs

Description

Programme

LS 5Correlative and multimodal microscopy

Appointment

Session chairs

Description

Programme

LS 5
Correlative and multimodal microscopy