Imaging of retina cellular and subcellular structures using ptychographic hard X-ray tomography.

Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200 nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray technique matched the electron microscopy data, demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200 nm, which remain available for further structural and biochemical investigations.

Correlation of live-cell imaging with volume scanning electron microscopy.

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi µ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods.

Three-dimensional mass density mapping of cellular ultrastructure by ptychographic X-ray nanotomography.

We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180 nm. As model for a biological system we use Chlamydomonas cells in buffer solution confined in a microcapillary. We use ptychographic X-ray computed tomography to image the entire specimen, including the 18 µm-diameter capillary, thereby providing directly an absolute mass density measurement of biological matter with an uncertainty of about 6%. The resulting maps have sufficient contrast to distinguish cells from the surrounding ice and several organelles of different densities inside the cells. Organelles are identified by comparison with a stained, resin-embedded specimen, which can be compared with established transmission electron microscopy results. For some identified organelles, the knowledge of their elemental composition reduces the uncertainty of their mass density measurement down to 1% with values consistent with previous measurements of dry weight concentrations in thin cellular sections by scanning transmission electron microscopy. With prospects of improving the spatial resolution in the near future, we expect that the capability of non-destructive three-dimensional mapping of mass density in biological samples close to their native state becomes a valuable method for measuring the packing of organic matter on the nanoscale.

FIB-SEM tomography in biology.

Three-dimensional information is much easier to understand than a set of two-dimensional images. Therefore a layman is thrilled by the pseudo-3D image taken in a scanning electron microscope (SEM) while, when seeing a transmission electron micrograph, his imagination is challenged. First approaches to gain insight in the third dimension were to make serial microtome sections of a region of interest (ROI) and then building a model of the object. Serial microtome sectioning is a tedious and skill-demanding work and therefore seldom done. In the last two decades with the increase of computer power, sophisticated display options, and the development of new instruments, an SEM with a built-in microtome as well as a focused ion beam scanning electron microscope (FIB-SEM), serial sectioning, and 3D analysis has become far easier and faster.Due to the relief like topology of the microtome trimmed block face of resin-embedded tissue, the ROI can be searched in the secondary electron mode, and at the selected spot, the ROI is prepared with the ion beam for 3D analysis. For FIB-SEM tomography, a thin slice is removed with the ion beam and the newly exposed face is imaged with the electron beam, usually by recording the backscattered electrons. The process, also called "slice and view," is repeated until the desired volume is imaged.As FIB-SEM allows 3D imaging of biological fine structure at high resolution of only small volumes, it is crucial to perform slice and view at carefully selected spots. Finding the region of interest is therefore a prerequisite for meaningful imaging. Thin layer plastification of biofilms offers direct access to the original sample surface and allows the selection of an ROI for site-specific FIB-SEM tomography just by its pronounced topographic features.

Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway.

The function of the liver is well-preserved during the aging process, although some evidence suggests that liver regeneration might be impaired with advanced age. We observed a decreased ability of the liver to restore normal volume after partial hepatectomy in elderly mice, and we identified a pathway that rescued regeneration and was triggered by serotonin. 2,5-dimethoxy-4-iodoamphetamine (DOI), a serotonin receptor agonist, reversed the age-related pseudocapillarization of old liver and improved hepatosinusoidal blood flow. After hepatectomy, the open fenestrae were associated with a restored attachment of platelets to endothelium and the initiation of a normal regenerative response, including the up-regulation of essential growth mediators and serotonin receptors. In turn, hepatocyte proliferation recovered along with regain of liver volume and animal survival. DOI operates through the release of VEGF, and its effects could be blocked with anti-VEGF antibodies both in vitro and in vivo. These results suggest that pseudocapillarization in the aged acts as a barrier to liver regeneration. DOI breaks this restraint through an endothelium-dependent mechanism driven by VEGF. This pathway highlights a target for reversing the age-associated decline in the capacity of the liver to regenerate.

Particle size and activation threshold: a new dimension of danger signaling.

Previous studies have shown that single-stranded RNA (ssRNA) mixed with protamine forms particles and activates immune cells through Toll-like receptors (TLRs). We have found that the size of protamine-RNA particles generated depends on the electrolyte content when mixing the 2 components. Moreover, we have evidenced that (1) nanometric particles induce production of interferon-alpha, whereas (2) micrometric particles mainly induce production of tumor necrosis factor-alpha (TNF-alpha) in human immune cells. We found that the mechanisms underlying these observations are (1) nanoparticles but not microparticles are selectively phagocytosed by plasmacytoid dendritic cells (pDCs), which produce interferon-alpha and (2) monocytes that produce TNF-alpha have a higher activation threshold than that of pDCs. Thus, at the same time as sensing pathogen-associated molecular patterns such as ssRNA, the immune system distinguishes the size of the associated structure in such a way as to trigger the adapted antivirus (nanometric) or antibacterial/antifungal (micrometric) immune response. Our results introduce a new dimension in danger signaling--how size qualitatively affects innate response.

Cyto- and hemocompatibility of a biodegradable 3D-scaffold material designed for medical applications.

In this study, the polyester urethane Degrapol (DP) was explored for medical applications. Electrospun DP-fiber fleeces were characterized with regard to fiber morphology, swelling, and interconnectivity of interfiber spaces. Moreover, DP was assayed for cell proliferation and hemocompatibility being a prerequisite to any further in vivo application. It was shown that DP-fiber fleeces produced at different humidity while spinning affects interconnectivity of interfiber spaces, such that the higher the humidity the looser the resulting fiber fleeces. When the spinning target was cooled with dry ice, the resulting DP-fibers remained less fused to each other. However, permeability for fluorescent beads was not significantly increased. Fibroblast adhesion and proliferation occurred in a comparable manner on native as well as on fibronectin or collagen I adsorbed DP-fiber fleeces. On DP-surfaces fibroblasts proliferated equally well as compared with glass or PLGA surfaces or DP-surfaces adsorbed with fibronectin or collagen I. In contrast, human umbilical vein endothelial cells proliferated only after adsorption of DP-surfaces with fibronectin or collagen I, indicating that different cell types respond differently to DP-surfaces. Furthermore, hemocompatibility of DP-surfaces was found to be similar or better to PLGA or stainless steel, both medically used materials. These experiments indicate that DP-fiber fleeces or surfaces might be useful for tissue engineering.

Engineered polyelectrolyte multilayer substrates for adhesion, proliferation, and differentiation of human mesenchymal stem cells.

Polyelectrolyte multilayer coatings have emerged as substrates to control cellular behavior, but interactions with human multipotent mesenchymal stromal cells (MSCs) have not been studied. We looked at layer-by-layer coatings of cationic poly-L-lysine (PLL) and anionic hyaluronic acid (HA) as substrates for MSCs of placenta and adipose tissue. This system allows for modulation of thickness (number of deposition cycles), stiffness (chemical cross-linking of bulk layer), and adhesiveness (fibronectin (FN) interface). Native, as-built PLL/HA multilayer coatings were poorly adhesive for MSCs despite spectroscopy-confirmed high surface density of pre-adsorbed FN. Stratification of cross-linked PLL/HA multilayers of different stiffnesses revealed that multilayers modified with a high cross-linking regimen became efficient substrates for MSC adhesion and proliferation. MSCs on cross-linked multilayers grew to confluence. Using comparative confocal microscopy analysis of PLL/HA multilayers with physically adsorbed versus chemically coupled FN, we demonstrated that cross-linking strongly influenced FN surface distribution, leading to denser presentation of adhesion sites for cells. The covalent affixation of FN promoted focal adhesion formation and was critical to maintaining densely grown MSC cultures over weeks for their differentiation. Multilayer-bound MSCs were capable of differentiating into osteocytes and chondrocytes upon culture with induction factors. Together, cross-linked, FN-terminated PLL/HA multilayers provide a versatile platform for studies of human MSCs for biotechnological or therapeutic applications.

Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nanoparticles.

Polycationic molecules form condensates with DNA and are used for gene therapy as an alternative to viral vectors. As clinical efficacy corresponds to cellular uptake, intracellular stability of the condensates, and bioavailability of the DNA, it is crucial to analyze uptake mechanisms and trafficking pathways. Here, a detailed study of uptake, stability, and localization of PLL-g-PEG-DNA nanoparticles within COS-7 cells is presented, using FACS analysis to assess the involvement of different uptake mechanisms, colocalization studies with markers indicative for different endocytotic pathways, and immunofluorescence staining to analyze colocalization with intracellular compartments. PLL-g-PEG-DNA nanoparticles were internalized in an energy-dependent manner after 2 h and accumulated in the perinuclear region after >6 h. The nanoparticles were found to be stable within the cytoplasm for at least 24 h and did not colocalize with the endosomal pathway. Nanoparticle uptake was approximately 50% inhibited by genistein, an inhibitor of the caveolae-mediated pathway. However, genistein did not inhibit gene expression, and PLL-g-PEG-DNA nanoparticles were not colocalized with caveolin-1 indicating that caveolae-mediated endocytosis is not decisive for DNA delivery. Clathrin-mediated endocytosis and macropinocytosis pathways were reduced by 17 and 24%, respectively, in the presence of the respective inhibitors. When cells were transfected in the presence of double and triple inhibitors, transfection efficiencies were increasingly reduced by 40 and 70%, respectively; however, no differences were found between the different uptake mechanisms. These findings suggest that PLL-g-PEG-DNA nanoparticles enter by several pathways and might therefore be an efficient and versatile tool to deliver therapeutic DNA.

Molecular design and characterization of the neuron-microelectrode array interface.

Electrophysiological activities of neuronal networks can be recorded on microelectrode arrays (MEAs). This technique requires tight coupling between MEA-surfaces and cells. Therefore, this study investigated the interface between DRG neurons and MEA-surface materials after adsorption of neurite promoting proteins: laminin-111, fibronectin, L1Ig6 and poly-l-lysine. Moreover, substrate-induced effects on neuronal networks with time were analyzed. The thickness of adsorbed protein layers was found between approximately 1 nm for poly-l-lysine and approximately 80 nm for laminin-111 on platinum, gold and silicon nitride. The neuron-to-substrate interface was characterized by Scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and SEM after in situ focused-ion-beam milling demonstrating that the ventral cell membrane adhered inhomogeneously to laminin-111 or L1Ig6 surfaces. Tight areas of 20-30 nm and distant areas <1 microm alternated and even tightest areas did not correlate with the physical thickness of the protein layers. This study illustrates the difficulties to predict cell-to-material interfaces that contribute substantially to the success of in vitro or in vivo systems. Moreover, focused ion beam (FIB)/SEM is explored as a new technique to analyze such interfaces.

Human preterm amnion cells cultured in 3-dimensional collagen I and fibrin matrices for tissue engineering purposes.

OBJECTIVE: In this study, human preterm amnion cells were investigated in 3-dimensional (3D) cell-matrix culture systems in an attempt to design therapeutic strategies for preterm premature rupture of the membranes. STUDY DESIGN: Three-dimensional collagen I and fibrin cell-containing biomatrices were created to mimic the architecture of native amnion. Amnion mesenchymal cells were embedded in 3D matrices, and epithelial cells were placed on top of these matrices. Cell viability and morphology were visualized by DiI-ac-LDL, F-actin, and nuclear staining. Proteolytic activity of matrix metalloproteinases (MMPs) was investigated using gelatine zymography. RESULTS: Preterm amnion epithelial and mesenchymal cells cultured in collagen I and fibrin matrices assume cell morphologies similar to those observed in vivo. Mesenchymal cells were capable of remodelling collagen I, as seen by extensive volume contraction, by 40% at day 1 and 80% at day 5. Matrix contraction was independent of the presence of epithelial cells, and could not be inhibited by GM6001 and/or aprotinin. No contraction was observed in fibrin matrices over 8 days. The migratory response of mesenchymal cells cultured in 3D fibrin matrices supplemented with fibronectin was associated with specific activated MMP-9. CONCLUSION: Three-dimensional fibrin matrices might be useful in amnion cell tissue engineering, including cell-matrix transplantation.

Mechanical properties, proteolytic degradability and biological modifications affect angiogenic process extension into native and modified fibrin matrices in vitro.

During initial stages of wound healing, fibrin clots provide a three-dimensional scaffold that induces cell infiltration and regeneration. Here, L1Ig6, a ligand for alphavbeta3 integrin was covalently incorporated within fibrin matrices to explore it as a matrix-immobilized angiogenic factor. Incorporation at concentrations greater than 1 microg/ml reduced the fibrin crosslink density, as reflected by measurements of elastic modulus and swelling. The influence of crosslink density on endothelial cell process extension was characterized by modulating factor XIII concentrations in the coagulation mixture. At low incorporated concentrations of L1Ig6, it was possible to compensate gel elastic modulus via increased factor XIII, but not at high concentrations of L1Ig6. Similar findings were found when matrix swelling was analyzed. Fibrin crosslink density strongly influenced endothelial cell process extension, fewer and shorter processes were observed at high crosslink density. Matrix metalloproteinases (MMPs) were required for process extension and zymography and Western blots identified MMP-2 but not MMP-9. The amount of active MMP-2 increased for endothelial cells cultured in native and L1Ig6-modified matrices or when stimulated with VEGF-A165. The data indicate that distinct matrix properties can be tailored such that they become biologically stimulating and respond to cellular proteolytic activities, being a prerequisite for potential use of such matrices in biomedical applications.

Spatial and temporal organization of adeno-associated virus DNA replication in live cells.

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.