Optical microscopy based on waveguide chips significantly reduces the complexity of the entire optical setup, enabling miniaturization by completely removing the excitation light path from the microscope. Instead, waveguides which tightly confine the guided light by total internal reflection due to a high refractive index contrast (HIC) to the surrounding media such as water and cells are used to deliver the illumination light to the sample. The evanescent field on top of the waveguide can be utilized for total internal reflection fluorescence (TIRF) excitation over an almost arbitrarily wide FOV that is intrinsically independent of the detection objective lens and in principle only limited by the waveguide design. Evanescent field excitation using waveguides was first introduced by Grandin et al. 17 , where a slab waveguide was used to generate an evanescent field over the large stretch of the waveguide chip. Slab waveguides (Fig. 1a) Here, we demonstrate waveguide chip-based super-resolution fluorescence imaging by two complementary approaches using ESI and dSTORM. The high intensity in the evanescent field generated by the HIC waveguide material is used for optical switching of fluorophores as required by dSTORM. In addition, the intrinsically multi-mode interference pattern within the waveguide is used to generate fluctuating intensity patterns for ESI. To demonstrate the applicability of waveguide chip-based super-resolution microscopy we visualize the connection of the actin cytoskeleton and plasma membrane fenestrations in liver sinusoidal endothelial cells (LSECs). RESULTS Chip-based single molecule localization microscopyThe performance of chip-based dSTORM is shown by imaging immunostained microtubules in rat LSECs 26 plated directly on the waveguide (Fig. 2a). Measuring the lateral profile along one straight microtubule filament reveals a hollow structure 27 which has been used earlier in localization microscopy as a benchmark sample [28][29][30] , discussed in detail in 31 . This shows a resolution of better than 50 nm ( Fig. 2b), confirmed by full-width-at-half-maximum (FWHM) values, localization precision 32 , and Fourier ring correlation 33,34 (FRC) calculations ( Supplementary Fig. 1). The resolution capability was further investigated by using DNA origami nanorulers that provide markers at (50 ± 5) nm distance as references. These can be clearly resolved in chip-based dSTORM (Fig. 2c,d, Supplementary Fig. 2) which shows a comparable performance to the widely used architecture of an inverted TIRF dSTORM setup (Fig. 2c,d, Supplementary Fig. 3).As an advantage over conventional setups, waveguide chip-based nanoscopy greatly benefits from the fact that the fluorescence excitation is independent of the detection objective lens. As fluorescence is excited by the evanescent field of the waveguide, the technique provides optical sectioning and excellent signal to background ratios at penetration depths below 200 nm ( Supplementary Fig. 4, Supplementary Fig. 5, Video 1), similar to objective-based TIRF...

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

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.

Background: Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared.

Background & Aims: Liver fibrogenesis -scarring of the liver that can lead to cirrhosis and liver cancer -is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs.Material and methods: Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR.Results: We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiationassociated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis.Conclusions: Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.

Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional nanoscopy lacks the ability to generate high throughput data, as the imaged region is small. Photonic chip-based nanoscopy has demonstrated the potential for imaging large areas, but at a lateral resolution of 130 nm. However, all the existing super-resolution methods provide a resolution of 100 nm or better. In this work, chip-based nanoscopy is demonstrated with a resolution of 75 nm over an extraordinarily large area of 0.5 mm x 0.5 mm, using a low magnification and high N.A. objective l ens. Furthermore, the performance of chip-based nanoscopy is benchmarked by studying the localization precision and illumination homogeneity for different waveguide widths. The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin ( L-SIGN) (or C-type lectin domain family member M ( CLEC4M)), mannose receptor C-Type 1 ( MRC1), legumain ( LGMN), G protein-coupled receptor 182 ( GPR182), Plexin C1 ( PLXNC1), and solute carrier organic anion transporter family member 2A1 ( SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint. NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.