A Joint Action in Times of Pandemic: The German BioImaging Recommendations for Operating Imaging Core Facilities During the SARS-Cov-2 Emergency.

Operating shared resource laboratories (SRLs) in times of pandemic is a challenge for research institutions. In a multiuser, high-turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could be operated with minimal risk of spreading SARS-CoV-2 between users and staff. Here, we describe the resulting guidelines and explain their rationale, with a focus on separating users in space and time, protective face masks, and keeping surfaces virus-free. These recommendations may prove useful for other types of SRLs. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Estimating hemodynamic shear stress in murine peripheral collateral arteries by two-photon line scanning.

Changes in wall shear stress of blood vessels are assumed to be an important component of many physiological and pathophysiological processes. However, due to technical limitations experimental in vivo data are rarely available. Here, we investigated two-photon excitation fluorescence microscopy as an option to measure vessel diameter as well as blood flow velocities in a murine hindlimb model of arteriogenesis (collateral artery growth). Using line scanning at high frequencies, we measured the movement of blood cells along the vessel axis. We found that peak systolic blood flow velocity averaged 9 mm/s and vessel diameter 42 µm in resting collaterals. Induction of arteriogenesis by femoral artery ligation resulted in a significant increase in centerline peak systolic velocity after 1 day with an average of 51 mm/s, whereas the averaged luminal diameter of collaterals (52 µm) changed much less. Thereof calculations revealed a significant fourfold increase in hemodynamic wall shear rate. Our results indicate that two-photon line scanning is a suitable tool to estimate wall shear stress e.g., in experimental animal models, such as of arteriogenesis, which may not only help to understand the relevance of mechanical forces in vivo, but also to adjust wall shear stress in ex vivo investigations on isolated vessels as well as cell culture experiments.

Tracking Microscope Performance: A Workflow to Compare Point Spread Function Evaluations Over Time.

Routine system checks are essential for supervising the performance of an advanced light microscope. Recording and evaluating the point spread function (PSF) of a given system provides information about the resolution and imaging. We compared the performance of fluorescent and gold beads for PSF recordings. We then combined the open-source evaluation software PSFj with a newly developed KNIME pipeline named PSFtracker to create a standardized workflow to track a system's performance over several measurements and thus over long time periods. PSFtracker produces example images of recorded PSFs, plots full-width-half-maximum (FWHM) measurements over time and creates an html file which embeds the images and plots, together with a table of results. Changes of the PSF over time are thus easily spotted, either in FWHM plots or in the time series of bead images which allows recognition of aberrations in the shape of the PSF. The html file, viewed in a local browser or uploaded on the web, therefore provides intuitive visualization of the state of the PSF over time. In addition, uploading of the html file on the web allows other microscopists to compare such data with their own.

Maturation of Platelet Function During Murine Fetal Development In Vivo.

OBJECTIVE: Platelet function has been intensively studied in the adult organism. However, little is known about the function and hemostatic capacity of platelets in the developing fetus as suitable in vivo models are lacking. APPROACH AND RESULTS: To examine fetal platelet function in vivo, we generated a fetal thrombosis model and investigated light/dye-induced thrombus formation by intravital microscopy throughout gestation. We observed that significantly less and unstable thrombi were formed at embryonic day (E) 13.5 compared with E17.5. Flow cytometry revealed significantly lower platelet counts in E13.5 versus E17.5 fetuses versus adult controls. In addition, fetal platelets demonstrated changed activation responses of surface adhesion molecules and reduced P-selectin content and mobilization. Interestingly, we also measured reduced levels of the integrin-activating proteins Kindlin-3, Talin-1, and Rap1 during fetal development. Consistently, fetal platelets demonstrated diminished spreading capacity compared with adults. Transfusion of adult platelets into the fetal circulation led to rapid platelet aggregate formation even in young fetuses. Yet, retrospective data analysis of a neonatal cohort demonstrated no correlation of platelet transfusion with closure of a persistent ductus arteriosus, a process reported to be platelet dependent. CONCLUSIONS: Taken together, we demonstrate an ontogenetic regulation of platelet function in vivo with physiologically low platelet numbers and hyporeactivity early during fetal development shedding new light on hemostatic function during fetal life.

Multiphoton Microscopy of Nonfluorescent Nanoparticles In Vitro and In Vivo.

Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano-bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO2 ), and silica (SiO2 ) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon-induced luminescence decaying over time. TiO2 NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano-bio interactions not only for fluorescent but also for some types of nonfluorescent NPs.

Stably integrated and expressed retroviral sequences can influence nuclear location and chromatin condensation of the integration locus.

The large-scale chromatin organization of retrovirus and retroviral gene vector integration loci has attracted little attention so far. We compared the nuclear organization of transcribed integration loci with the corresponding loci on the homologous chromosomes. Loci containing gamma-retroviral gene transfer vectors in mouse hematopoietic precursor cells showed small but significant repositioning of the integration loci towards the nuclear interior. HIV integration loci in human cells showed a significant repositioning towards the nuclear interior in two out of five cases. Notably, repositioned HIV integration loci also showed chromatin decondensation. Transcriptional activation of HIV by sodium butyrate treatment did not lead to a further enhancement of the differences between integration and homologous loci. The positioning relative to splicing speckles was indistinguishable for integration and homologous control loci. Our data show that stable retroviral integration can lead to alterations of the nuclear chromatin organization, and has the potential to modulate chromatin structure of the host cell. We thus present an example where a few kb of exogenous DNA are sufficient to significantly alter the large-scale chromatin organization of an endogenous locus.

Chromosome territories--a functional nuclear landscape.

Understanding nuclear architecture is indispensable for understanding the cell-type-dependent orchestration of active and silent genes and other nuclear functions, such as RNA splicing, DNA replication and repair. Yet, while it is now generally agreed that chromosomes in the cell nucleus are organized as chromosome territories, present models of chromosome territory architecture differ widely with respect to the possible functional implications of dynamic changes of this architecture during the cell cycle and terminal cell differentiation.

The radial nuclear positioning of genes correlates with features of megabase-sized chromatin domains.

A nonrandom radial nuclear organization of genes has been well documented. This study provides further evidence that radial positioning depends on features of corresponding ∼1 Mbp chromatin domains (CDs), which represent the basic units of higher-order chromatin organization. We performed a quantitative three-dimensional analysis of the radial nuclear organization of three genes located on chromosome 1 in a DG75 Burkitt lymphoma-derived cell line. Quantitative real-time polymerase chain reaction revealed similar transcription levels for the three selected genes, whereas the total expression strength (TES) calculated as the sum of transcription of all genes annotated within a surrounding window of about 1 Mbp DNA differed for each region. Radial nuclear position of the studied CDs correlated with TES, i.e., the domain with the highest TES occupied the most interior position. Positions of CDs with stable TES values were stably maintained even under experimental conditions, resulting in genome-wide changes of the expression levels of many other genes. Our results strongly support the hypothesis that knowledge of the local chromatin environment is essential to predict the radial nuclear position of a gene.

Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation.

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

Mediator subunits: gene expression pattern, a novel transcript identification and nuclear localization in human endothelial progenitor cells.

Mediator of RNA polymerase II transcription subunits (MEDs) serve to promote the assembly, activation, and regeneration of transcription complexes on core promoters during the initiation and re-initiation phases of transcription. There are no studies on the Mediator complex during development of endothelial progenitors (EPCs). Here, we have analysed all known MEDs during the differentiation of EPCs, by expression profile studies at RNA level and, for a limited subset of MED subunits, also at protein level. Since beneficial effects of L-arginine on EPCs have been described, we have also examined its effect on the expression of Mediator subunit coding genes. Through RT-PCR we have found increased expression for MED12 and decreased levels for MED30 after l-arginine treatment; Western blot analysis do not agree entirely with the RNA data in the identification of a putative protein product. Furthermore, we have analysed the three-dimensional nuclear positions of MED12 and MED30 genes in the presence of l-arginine treatment. Our major finding is the identification of a novel transcript of MED30, termed MED30 short (MED30s) generating by alternative splicing. Our results showed that the mRNA of this novel isoform is present only in circulating cells, but it is not expressed in cultured adherent cells. These findings are broadly relevant and will contribute to our understanding of the role of Mediator in eukaryotic gene expression. Despite the need to confirm the in vivo presence of the protein of this novel isoform, the presence of this novel RNA raises the possibility of regulating pathophysiological mechanism in progenitors.

BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

Sphingosine-1-Phosphate Attenuates Lipopolysaccharide-Induced Pericyte Loss via Activation of Rho-A and MRTF-A.

The high mortality seen in sepsis is caused by a systemic hypotension in part owing to a drastic increase in vascular permeability accompanied by a loss of pericytes. As has been shown previously, pericyte retention in the perivascular niche during sepsis can enhance the integrity of the vasculature and promote survival via recruitment of adhesion proteins such as VE-cadherin and N-cadherin. Sphingosine-1-phosphate (S1P) represents a lipid mediator regulating the deposition of the crucial adhesion molecule VE-cadherin at sites of interendothelial adherens junctions and of N-cadherin at endothelial-pericyte adherens junctions. Furthermore, in septic patients, S1P plasma levels are decreased and correlate with mortality in an indirectly proportional way. In the present study, we investigated the potential of S1P to ameliorate a lipopolysaccharide-induced septic hypercirculation in mice. Here we establish S1P as an antagonist of pericyte loss, vascular hyperpermeability, and systemic hypotension, resulting in an increased survival in mice. During sepsis S1P preserved VE-cadherin and N-cadherin deposition, mediated by a reduction of Src and cadherin phosphorylation. At least in part, this effect is mediated by a reduction of globular actin and a subsequent increase in nuclear translocation of MRTF-A (myocardin-related transcription factor A). These findings indicate that S1P may counteract pericyte loss and microvessel disassembly during sepsis and additionally emphasize the importance of pericyte-endothelial interactions to stabilize the vasculature.

Parental genomes mix in mule and human cell nuclei.

Whether chromosome sets inherited from father and mother occupy separate spaces in the cell nucleus is a question first asked over 110 years ago. Recently, the nuclear organization of the genome has come increasingly into focus as an important level of epigenetic regulation. In this context, it is indispensable to know whether or not parental genomes are spatially separated. Genome separation had been demonstrated for plant hybrids and for the early mammalian embryo. Conclusive studies for somatic mammalian cell nuclei are lacking because homologous chromosomes from the two parents cannot be distinguished within a species. We circumvented this problem by investigating the three-dimensional distribution of chromosomes in mule lymphocytes and fibroblasts. Genomic DNA of horse and donkey was used as probes in fluorescence in situ hybridization under conditions where only tandem repetitive sequences were detected. We thus could determine the distribution of maternal and paternal chromosome sets in structurally preserved interphase nuclei for the first time. In addition, we investigated the distribution of several pairs of chromosomes in human bilobed granulocytes. Qualitative and quantitative image evaluation did not reveal any evidence for the separation of parental genomes. On the contrary, we observed mixing of maternal and paternal chromosome sets.

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

BACKGROUND: von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. METHODS AND RESULTS: The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. CONCLUSION: CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

Three-dimensional positioning of genes in mouse cell nuclei.

To understand the regulation of the genome, it is necessary to understand its three-dimensional organization in the nucleus. We investigated the positioning of eight gene loci on four different chromosomes, including the beta-globin gene, in mouse embryonic stem cells and in in vitro differentiated macrophages by fluorescence in situ hybridization on structurally preserved nuclei, confocal microscopy, and 3D quantitative image analysis. We found that gene loci on the same chromosome can significantly differ from each other and from their chromosome territory in their average radial nuclear position. Radial distribution of a given gene locus can change significantly between cell types, excluding the possibility that positioning is determined solely by the DNA sequence. For the set of investigated gene loci, we found no relationship between radial distribution and local gene density, as it was described for human cell nuclei. We did find, however, correlation with other genomic properties such as GC content and certain repetitive elements such as long terminal repeats or long interspersed nuclear elements. Our results suggest that gene density itself is not a driving force in nuclear positioning. Instead, we propose that other genomic properties play a role in determining nuclear chromatin distribution.

Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining.

BACKGROUND: GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH). Direct comparisons of these detection methods are scarce, however. RESULTS: We provide a quantitative comparison of all three approaches. We make use of a cell line that contains a transgene array of lac operator repeats which are detected by GFP-lac repressor fusion proteins. Thus we can detect the same structure in individual cells by GFP fluorescence, by antibodies against GFP and by FISH with a probe against the transgene array. Anti-GFP antibody detection was repeated after FISH. Our results show that while all four signals obtained from a transgene array generally showed qualitative and quantitative similarity, they also differed in details. CONCLUSION: Each of the tested methods revealed particular strengths and weaknesses, which should be considered when interpreting respective experimental results. Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.

MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse.

BACKGROUND: Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. RESULTS: All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. CONCLUSION: Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

Two-photon microscopy allows imaging and characterization of cochlear microvasculature in vivo.

Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanate- or Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of 90.0±22.7 µm. Imaging yielded a mean contrast-to-noise ratio (CNR) of 3.3±1.7. Mean diameter in vivo was 16.5±6.0 µm for arterioles and 8.0±2.4 µm for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with 12.2±1.6 µm and 6.6±1.0 µm, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P<0.001 and P=0.022, two-way ANOVA). Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research.

Thymosin ß4 attenuates microcirculatory and hemodynamic destabilization in sepsis.

OBJECTIVE: The actin polymerization regulator Thymosin ß4 (Tß4) has been shown to be involved in angiogenesis, wound healing, cell survival and anti-inflammatory responses. We have previously shown that Tß4 is capable of recruiting pericytes, thus stabilizing the endothelial barrier function. Here, we analyzed whether treatment with Tß4 is able to reduce the pericytes loss in lipopolysaccharides (LPS)-induced sepsis and to improve the hemodynamic function and survival in C57BL/6 mice. METHODS: Fourteen days before LPS injection, the mice were injected with an adeno-associated virus carrying the Tß4 (rAAV.Tß4) or LacZ gene (rAAV.LacZ). A sepsis-severity score was assessed, and non-invasive hemodynamic and permeability measurements were performed. Heart and muscle samples were analyzed for PECAM-1(+) capillaries and NG2(+)pericytes. RESULTS: At 36 h, there was a decrease of sepsis severity score in rAAV.Tß4-treated animals as compared to rAAV.LacZ-treated control. rAAV.Tß4-treated animals displayed lower perivascular leakage and higher blood pressure compared to control. Of note, the rAAV.Tß4 group showed a higher pericyte count in heart and peripheral muscle samples. Finally, Tß4-treatment reduced mortality compared to control. CONCLUSION: The data indicate a preventive role of Tß4 in septic hypercirculation and highlight Tß4 as a potential therapeutic target in severe sepsis.