Microscopy Conference 2023

Prof.

Session chairs

Prof. Dr.

PD Dr.

STEM-in-SEM of biological specimens in correlation with TEM

Abstract text (incl. figure legends and references) 1. Introduction Scanning transmission electron microscopy in scanning electron microscopes (STEM-in-SEM) at low primary electron energies (≤ 30 keV) yields a pronounced image contrast for weakly scattering biological materials [1] and provides a complementary technique to transmission electron microscopy (TEM) at high electron energies. SEMs are widely available and can be equipped with a STEM detector. Further advantages compared to TEM are reduced maintenance costs and less demanding alignment procedures. STEM-imaging can be combined with secondary electron imaging, rendering STEM-in-SEM a multimodal technique for imaging both surface and subsurface features [2]. 2. Objectives In this work, the competitiveness of STEM-in-SEM as an alternative to TEM is explored. The two techniques are compared with respect to their resolution and signal-to-noise ratio. 3. Materials and Methods Thin microtome slices of A549 lung carcinoma cells exposed to silica nanoparticles (NPs) are used for 200 keV TEM imaging in a Philips CM200. Bright-field (BF) STEM-in-SEM images are taken from the same region of interest in a FEI Helios G4 FX at 30 keV. The two techniques are compared by evaluating the spatial resolution and the contrast-to-noise-ratio CNR = (S-B)/σB (S: signal, B: background intensity, σB: standard deviation of B) of the images. As the contrast in TEM depends also on defocus and aperture settings, the beam convergence and detection angles for the images in Figure 2 are chosen such that similar image signals can be expected due to the reciprocity theorem for STEM and TEM [3]. 4. Results Figure 1 shows images of the same region of a cell section with NPs. To improve the contrast, the TEM image (Figure 1a) was taken with a defocus 1.5 μm. A line scan over a small feature reveals a feature size of 2 nm, estimated by the full-width-at-half-maximum (FWHM). The same feature in the BF-STEM image (Fig. 1b) also shows a resolution of 2 nm. The evaluation of the CNR is shown in Figure 2. A line scan through one of the dark particles reveals a more pronounced noise in the TEM image (Figure 2a) with CNR=3. The 30 keV BF-STEM image in Figure 1b shows a reduced noise with CNR=20. 4. Conclusions STEM-in-SEM is a powerful technique for analyzing biological specimens, combining good CNR with a high spatial resolution. The resolution is sufficient for many applications making STEM-in-SEM well competitive to TEM. Figure 1. Spatial resolution of 2 nm estimated by the FWHM of a linescan over a small feature. a) TEM image with a defocus of 1.5 µm. b) BF-STEM image of the same region. Figure 2. Line scan over a NP. a) CNR=3 for the TEM image. b) CNR=20 for the BF-STEM image [1] U Kaiser et al., Ultramicroscopy 111 (2011), p. 1239. doi: 10.1016/j.ultramic.2011.03.012 [2] C Sun et al., J Mater Sci 55 (2020), p. 13824. doi: 10.1007/s10853-020-04970-3 [3] CB Carter and DB Williams, "Transmission Electron Microscopy", Plenum Press, New York, 1996

LS 5: Correlative and multimodal microscopy

LS 6: Pathology, pathogens and diagnostics

Poster

Multiscale imaging pipeline integrating x-ray tomography, classical histology, and SEM to target sub-volumes in conventional paraffin samples

Abstract text (incl. figure legends and references) Introduction: Paraffin samples still remain the primary source of pathological diagnosis and for understanding disease development and progression. Due to the vast amount of paraffin samples available to scientific laboratories and universities around the world [1], we need to rethink modalities for imaging targeted structures that go beyond the limits of standard light microscopy (LM) and that achieve higher resolution. Objectives: Our goal is to develop a correlated workflow for targeting volumes of interest within a standard paraffin block. Starting with X-ray based imaging to create a reference volume, we then combine LM and SEM imaging to address specific subvolumes. Materials &amp; Methods: Samples - Tissue from human lungs affected by COVID-19 was fixed in formalin and embedded in paraffin (FFPE) according to standard protocols. X-ray microscopy - Entire paraffin blocks were μCT-imaged (Phase Contrast CT) using a synchrotron radiation-based system (SRCT, Synchrotron Elettra, Trieste), initially with an isometric voxel size of 4μm. Based on the 3D scan of the entire block, targeted punch biopsies (cylinders with a diameter of 4 mm) were taken and scanned again with a voxel size of 2μm. Histology - Serial sections (5μm thick) were cut from a selected volume, stained (AB-PAS) and imaged using digital LM (slide scanner). Electron microscopy - Sections were stained with 3% uranyl acetate and lead citrate or/and sputtered with 3nm of Pt/Pd (80/20%). SEM (Zeiss Ultra 55) analysis was performed on regions pre-defined in the LM scan. Integration – To adjust possible deformations of the sections due to LM/SEM preparation, the data was registered with ,,Fuxlastix'' [2] Results: Comparison of a virtual slice from an X-ray volume (fig 1A) with a physical section cut from the corresponding paraffin block and stained for glycoproteins (fig 1B) shows perivascular fibrotic tissue formation in both modalities. SEM imaging (fig 1C, D) of regions defined in the LM scan illustrates the amount of detail obtainable at high resolution. By integrating the LM and SEM data into the X-ray volume, it is possible to identify and segment extended extracellular matrix elements such as the elastin cables (yellow in fig 2B &amp; C) within the volume. Combining a cross-section of an alveolar tip (insets fig 2A) highlighted in purple in the X-ray volume (fig 2 B top) with the segmented elastin cables (fig 2B bottom) indicates where the elastin cables &quot;penetrate&quot; the alveolar tip. Conclusion: The use of X-ray tomography for non-destructive targeting is a goal-oriented method. We have developed a consistent correlative workflow for imaging paraffin samples in order to go beyond the restricted spatial resolution offered by traditional LM analysis. Furthermore, by using SRCT-based segmentations, we are able to make interpretations of their 3D context and represent them ultrastructurally by imaging the slice in the SEM. [1] L Cheng, DG Bostwick, (Eds.) - Essentials of Anatomic Pathology, Humana Press, 2002 [2] J Albers et al. - Elastic transformation of histological slices allows precise co‐registration with microCT data sets for a refined virtual histology approach, Scientific Reports, 2021

Magnetic resonance microscopy for the 3D-non-destructive analysis of the tendon micro-structure under strong mechanical load supported by polarization sensitive optical microscopy and SEM data

Abstract text (incl. figure legends and references) 1 Introduction Magnetic Resonance Microscopy (MRM) is limited in spatial resolution (a≈10µm) and sensitivity compared to optical microscopy (OM) or SEM but offers the advantages of being non-destructive, 3-Dimensional imaging of intact 3D biological tissue and a multifold variety of different contrast weightings (eg. T2/T1, magn. susceptibility, diffusivity, chemical information). These features qualify MRM for investigating biological samples and delineation of microstructural changes as a consequence of biological or mechanical impact. The elementary functional element of the tendon is represented by collagen filaments. These are arranged to larger fiber bundles, surrounded by the MR-bright endotenon, in a hierarchical, self-similar way from primary to tertiary fiber bundles1. Strong mechanical load without rupture, e.g. by abrupt trauma in sport or repetitive dynamic fatigue overload might change the microstructural arrangement of fiber bundles permanently and therefore might result in increased risk for permanent fatigue aches and even rupture within routine sport activities. 2 Objectives .1 Can high resolution MRM delineate permanent changes in the microstructure of tendons as a consequence of strong mechanical load? .2 What are these microstructural changes and can they be related to the results of ex-vivo investigations on human tendons using OP or SEM? What bio-physical model could be used for the interpretation of the observed changes in T2* contrast MRM? 3 Materials/Methods 3.1 MR-microscopy The spatial encoding in 2DFT-MR-imaging is obtained via slice selective excitation, phase- and frequency encoding of the sample emitted radiofrequency (rf) signal. The extraordinary high microscopic spatial resolution on an Ultra-High-Field (B=7T) human MR-scanner was achieved using a prototype strong magn. field gradient insert with sensitive rf-detectors2. 2D and 3D- T2*-weighted images (VS: 64x64x60µm3) were obtained with short encoding time TE for visualization of tendon tissue with short relaxation time T2*. 3.2 Tendon-tissue samples Several samples (l≈5cm) from cattle Achilles tendon were halved into two parts; one segment was exposed to a mechanical load (F≈600N). The samples were scanned with orientation of the main tendon axis and mechanical load parallel to the outer static magnetic field B of the MR-scanner. 4 Results An MR-microscopic axial slice out of a Multi-slice MR data set (fig.1) and a sagittal view reconstructed from a high-resolution isotropic 3D data set are shown exemplarily below (fig.2). The MRM images were compared to polarization sensitive OM and SEM of the rat tendon under mechanical load3. The main direction of tendon fiber filaments changes for a length of about 60µm (crimp structure of tendon) until being oriented back again. The number and angle of this crimp misalignment reduces with load (flattening triangle model) and was also observed in MRM (disappearing of the hyperintense filaments) as a consequence of the Magic angle effect4 in MR. 5 Conclusion Advanced MR-technology based on prototype insert hardware on UHF scanners is capable of the non-destructive visualization of permanent changes in the microstructure of tendons with load below rupture level. The MR appearance can be interpreted within a flattening alignment model of the tendon crimp structure. Thus, MR-microimaging offers perspectives for the early diagnosis of pre-damage to tendons and interpretation of undefined tendon aches.

Morphological evaluation of the stomach of the African giant-pouched rat (Cricetomys gambianus) using gross anatomy, histology and scanning electron microscopy

Abstract text (incl. figure legends and references) The African giant pouched-rat (AGR) is semi-fossorial, nocturnal and omnivorous wild rodent that is widely distributed in sub-Saharan Africa.. AGR is used to detect land mines and for tuberculosis investigation and other biomedical uses. The stomach plays important roles in the physical and digestive process. There are reports on gross anatomy, morphometric and histology of the digestive tract of AGR (Knight and Knight-Eloff; 1987). Detailed investigations on the internal morphology of the stomach of AGR are scant, and studies that used scanning electron microscopic technique (SEM) for investigation rare. This study therefore seeks to examine the relationship between stomach structure and food habits by gross dissection, stereomicroscopy, and histology and scanning electron microscopic techniques. Six adult rats of either sex captured from the wild at night with metal-cage traps were used. Grossly, the stomach externally showed a U-shaped compartmentalized parts with copious sacculations. Well developed oesophageal groove, numerous papillae (corpus of non-glandular zone) and pyloric pouch were identified. An internal limiting ridge demarcated glandular from non-glandular part (FIGS.1A-1D). The four regions of the glandular stomach are cardiac, fundus, pylorus and antrum, while non-glandular zone consists of coma-shaped fundus and out-pocketing saccus cecum. Internally non-glandular zones are comprised of protruded papillae-like structures and thick wall, the glandular part showed smooth fundic mucosa, pyloric sphincter and gastric diverticular folds. The mean weight and length and width of the stomach were 27.7g, 19.5 cm and 3.9 cm respectively. Histologically (Fig. 2A), the arrangement of stomach tissues was as seen in typical mammalian pattern with variations in the glandular and non-glandular compartments. The wall of the stomach in the glandular and non-glandular regions showed four layers; mucosa supported by lamina propria, submucosa (loose connective tissue, vessels and nerves), muscularis (inner oblique, inner circular and out longitudinal muscle layers) and serosa. Non-glandular corpus and fornix ventriculi was covered in the lumen by numerous papilla lined by stratified keratinized epithelium. The glandular part is folded into rugae and the complex epithelial/ glandular lining contains depressions (gastric pits) which provided entrance into gastric glands lined by five types of typical epithelial cells There were variations of these components in the parts of the glandular antrum, cardiac, fundic and pyloric glands. The presence of neutral and acidic mucins in the gastric pits was demonstrated by PAS and AB reactions. Under SEM, the mucosal surface of the non-glandular compartment showed projections of robust papillae with longitudinal invaginations and desquamated keratinous structures of the papillae(Figs.2B-D). In fundic glandular part, gastric plicae with numeous openings of gastric glands were apparent and several longitudinal rigdes were seen amongst the plicae in the pyloric antrum. The study demonstrated that the stomach of AGR is complex as in other Cricetids (Perrin and Kokkin, 1986). The presence of sacculations, well-developed, numerous papillae, and pyloric pouch makes it remarkably different from other wild rats and supports its omnivorous dietary habit and symbiotic bacteria role in the stomach. Knight &amp; Knight-Eloff (1986). J. Zool., 213:7-22 Perrin &amp; Kokkin (1986). South Afr. J. Zool., 21(3):202-210

Micro- and nanoplastics contamination detection in plant samples by correlative microscopy

Abstract text (incl. figure legends and references) Correlative light electron microscopy (CLEM) has been used for the detection of plastics pollution with plants. Entries of the ubiquitous world-wide plastics pollution into nature thus enter the human food chain as degradation products, influencing environmental habitat and health. Micro- and nanoplastics as foreign bodies contaminating beverages and foods are likely to have a long-term impact on nutrition growth, public and personal health. Nanoparticles and their spatial location are generally difficult to detect in biological specimens, especially plastic particles due to their analogous hydrocarbon-based matrix. Reliable analysis methods for the discovery of micro- and nanoplastic particles in entities are not fully established yet. We determined that light microscopy (LM) is usable for a coarse estimation of the contamination grade of plant samples. Light microscopy, due to its optical diffraction limit, rather detects nanoparticle patches than single particles, shown using fluorescent polymer nanoparticles. Scanning electron microscopy (SEM) instead provides higher resolution, especially for single particle detection. But it requires extended EM sample staining preparation to depict hydrocarbon-based specimens. Using both microscopy types in a CLEM approach with TESCAN CORAL, the necessary insights to the contaminated plant areas are provided with single nanoparticle resolution. It reveals that nanoparticles seem to be tightly attached to the plant material (cf. Fig. 1). It indicates further that basic food cleaning procedures might be insufficient for full particle removal. The presented approach circumvents correlative topography issues of the 3D-plant samples. CLEM provided a fast and reliable analysis method for the semiquantitative analysis &amp; detection of micro- and nanoparticles plant contamination. (Fig.1: Nanoplastics particles attached to the leaf surface)

LS 7: Advances in sample preparation

Contextual interactive light and electron microscopy

Abstract text (incl. figure legends and references) Introduction: In biological context subcellular activity always shares both structural and functional components. In consequence, the examination of a biological function will gain benefit from a spatially resolved image of the location of activity. In turn, the interpretation of biological patterns is substantially supported by information regarding the function expected. This is commonly addressed by recording images of different spatial resolution and different functional information values. A combination of both different image types makes it possible to superpose function and location. We remove this process of superposition by spanning up a common functional and spatial context and interpret the electron microscopy records using the help of single molecule light microscopy-derived functional information and subsequent topology analysis. Objective: The approach sketched here is based on the assumption that functionality and spatial organization are coupled if a common size regime is covered. This assumption makes it possible to us to follow a biological function inside a native environment with a resolution nearby the one typical for electron microscopy. We subsequently interpret the higher resolved electron microscopic recording in terms of the functional information derived from the first part of the examination. Material &amp; Methods: The selection of a biological function of interest is done via conventional immunostaining with fluorescently and gold labelled antibodies. The specimens are recorded by single molecule light microscopy (SMLM). We record the dye molecule blinking signals and the gold mediated fluorescence. The resulting signal matrix is not (only) converted into an image but also a template for topology based analyses. Subsequent to this first procedure to extract biological function, the specimen is stained by ruthenium oxide. The large difference in atomic mass between ruthenium and gold makes it possible to identify the locus of the function stained via fluorescent antibodies inside the spatial organization as given by the ruthenium stain. Results: We localize the biological function inside an environment contrasted by heavy metal staining. In difference to conventional immune-gold applications the electron microscopy image is interpreted by a set of information derived from the same specimen but not the same locality. The presence of gold makes it possible to coordinate this independent information in a common functional space inside a cell. The topology analyses do not improve the spatial resolution of the electron microscopy imaging but complement it with functional information. Discussion: With our approach we span a common context between single molecule light microscopy and electron microscopy. This context makes it possible to interpret the appearance of an electron microscopic record under terms of the biological function supported by the electron microscopic derived structure. For instance, the information regarding spatial organization derived from topology analysis can give a reference parameter to estimate whether the electron microscopy imaged pattern is a true basis for biological function or a superposition of original biological structure and preparation mediated artifacts.

Comparative morphological features of the stomach (Proventriculus and Gizzard) in two species of Columbidae (Red-eyed turtle dove: Stretopelia semitoquarta; Domestic pigeon: Columba livia domestica) in the wild and domestic environment

Abstract text (incl. figure legends and references) INTRODUCTION: Among the different bird species, the shape, size and structure of the stomach varies with diet (Duke, 1997). The avian stomach is composed of two definite compartments separated by a constriction: the cranial glandular proventriculus (PV) and a larger caudal non-glandular gizzard (GZ). The stomach of flesh-eating species of birds is primarily a storage organ appropriate for chemical digestion of soft diet. But, the stomach of herbivorous and omnivorous birds is adapted for mechanical degradation of tougher food materials through strong muscular development of the stomach (King and McLelland, 1975). The red-eyed dove (RD) and domestic pigeon (DP) belong to the Columbidae which feed primarily on seeds, fruits, and plants. But the domestic and wild feeding habitats of the species could influence their granivorous and omnivorous habits and subsequently impact on the structure of the stomach. Large-sized grains were specifically recovered from stomach of RD unlike the small-sized grains from the DP. Studies have been carried out on the histology of the proventriculus and gizzard in many species especially poultry (fowl, guinea fowl and turkeys, quail) and few feral species (common starlings, hooded crow and Australian passerines). Few reports have described the stomach linings of the Columbidae especially wild doves by ultrastructural evaluation. The present investigation seeks to evaluate the structural features of the proventriculus and gizzard in the wild RD and DP and related the features with dietary habits. METHODOLOGY: The work used gross dissection, stereomicroscopy, histology and scanning electron microscopy following routine protocols. Six adult RD (body weight: 148.69g) and 6 DP (body weight: 293.56g) birds of either sex were used. Humane handling and cervical dislocation for euthanasia were used. RESULTS: Gross results showed that the relationship of the stomach to the left lobe of liver, duodenum, spleen and abdominal air sacs was similar in both species. Grossly typical features of cone-shaped PV and lens-shaped GZ were present (Figs 1a-e). It was located within intestinal peritoneal sac. The total GIT weight and stomach weight for were 12.06g, 27.61g and 5.12g, 8.51g respectively for RD and DP. The stomach represented 41.4% and 30.1% of the GIT in RD and DP respectively. It showed 3.4% and 2.8% of body weight in RD and DP respectively. Under SEM (Figs 2a-f), PV formed spiral folds (plicae) around a central glandular opening and the number and anastomotic arrangement varied in the species. The glandular openings were surrounded by longitudinal and transverse mucosal folds of the epithelium. Evidence of secretory activity of mucin-like substances was apparent. Surface structures resembling variably shaped projections of membrane were in the gizzard of the birds by SEM. Histologically (Figs. 2g-i), four typical tunics were represented in the two species. Large proventricular glands were tubuloalveolar lobules with secretory ducts and arranged were in a definite row and shape in the muscularis mucosa. The koilin membrane and tunica muscularis of GZ was thicker in the RD. Simple tubular glands were with the thick muscular layers. CONCLUSION:The morphological features of the stomach did not differ significantly in the two birds indicating convergence of dietary components. King and Mclelland (1975). Outlines of Avian Anatomy, Bailliere Tindall, London Duke (1997). Proc. Nurt. Soc., 56, 1049-1056.

Fluorescent dyes for in-resin CLEM with conventional EPON embedding

Abstract text (incl. figure legends and references) Introduction In-resin correlative light and electron microscopy (CLEM) facilitates a high-precision registration of the fluorescence signal upon the EM image. Current approaches most often employ acrylic resins, such as Lowicryl HM20, for preserving fluorescence while compromising the ultrastructural preservation (Buerger et al., 2021). Epoxy resins, such as EPON 812, on the other hand are the optimal choice for preserving ultrastructure. However, embedding samples in epoxy resins leads to a reduction in antigenicity and fluorescence. Nevertheless, it was shown that on-section CLEM following a conventional sample preparation including chemical fixation, post-fixation with osmium tetroxide, dehydration at room temperature and embedding in epoxy resins is possible with partly specifically adapted fluorescent proteins or protein tags labelled with certain dyes (Müller et al., 2017; Peng et al., 2022). Objectives We set out to further test the broad applicability of different fluorescent dyes in a conventional in-resin CLEM workflow with embedding in EPON. We plan to demonstrate the robustness of this by application to different cellular organelles, which allows us to achieve a high-precision registration with minimal effort in terms of sample preparation. Materials &amp; methods Cell lines and cell culture HeLa cells expressing the HaloTag fused to various markers were labelled with different fluorescent ligands, imaged by confocal laser scanning microscopy (cLSM), if needed, and further processed for EM. Sample preparation for on-section CLEM Cells were fixed with 2.5 % glutaraldehyde and post-fixed for 30 min using 1 % OsO4 and 1.5 % K4[Fe(CN6)] on ice. Samples were dehydrated and embedded in EPON. After polymerization, 250 nm thin sections were cut, collected on mesh grids and imaged at a cLSM. Results The fluorescence of mitochondria, Golgi apparatus and Salmonella effector proteins labelled with TMR or JFX554 is preserved after conventional sample preparation and embedding in EPON. Conclusion The in-resin CLEM workflow devised in this study offers a very promising tool for cases in which Lowicryl embedding cannot or does not need to be used, but a registration of the LM to the EM image with high accuracy is desirable. Live-cell imaging and direct fixation on the LM stage can be easily integrated. References Buerger, K., Schmidt, K.N., Fokkema, J., Gerritsen, H.C., Maier, O., de Vries, U., Zaytseva, Y., Rachel, R., and Witzgall, R. (2021). Chapter 8 - On-section correlative light and electron microscopy of large cellular volumes using STEM tomography. In Methods Cell Biol, T. Müller-Reichert, and P. Verkade, eds. (Academic Press), pp. 171-203. Müller, A., Neukam, M., Ivanova, A., Sönmez, A., Münster, C., Kretschmar, S., Kalaidzidis, Y., Kurth, T., Verbavatz, J.-M., and Solimena, M. (2017). A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags. Scientific Reports 7, 23. Peng, D., Li, N., He, W., Drasbek, K.R., Xu, T., Zhang, M., and Xu, P. (2022). Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM. Cells 11, 1077. Figure Legends Fig. 1: Conventional in-resin CLEM with EPON embedding of mitochondria labelled with fluorescent dyes. Fluorescence of Tom20-HaloTag stained with JaneliaFluorX 554 (A-C) or TMR (D-F) is preserved after conventional embedding in EPON. Scale bars: 2 µm.

PS 15
Poster session LS 5: Correlative and multimodal microscopy

Termin

Session chairs

Programm

PS 15Poster session LS 5: Correlative and multimodal microscopy

Termin

Session chairs

Programm

PS 15
Poster session LS 5: Correlative and multimodal microscopy