Marlene Thaler

Characterization of the stem cell system of the acoel Isodiametra pulchra

BackgroundTissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration.ResultsWe established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation.ConclusionsWe introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.

Visualization and analysis of superparamagnetic iron oxide nanoparticles in the inner ear by light microscopy and energy filtered TEM

UNLABELLED Nanoparticles as potential carriers for local drug transfer are an alternative to systemic drug delivery into the inner ear. We report on the first in vitro tests of a new ferrogel consisting of superparamagnetic iron oxide nanoparticles (SPIONs) and a Pluronic(®) F127 (PF127) copolymer. Pluronic copolymers possess a unique viscosity-adjustable property that makes PF127 gels easy to handle compared to conventional cross-linked hydrogels. This ferrogel was successfully tested in cadaver human temporal bones as well as in organotypic explant cultures of mouse inner ears. SPIONs were identified by light microscopy and localized with different imaging modes in energy-filtered transmission electron microscopy. Our approach shows a promising possibility to use iron oxide nanoparticles, which are suitable for visualization and characterization at both the light- and electron-microscopic levels. FROM THE CLINICAL EDITOR The authors report the first in vitro tests of a new ferrogel consisting of superparamagnetic iron oxide nanoparticles (SPIONs) and a Pluronic® F127 (PF127) copolymer for drug delivery in the inner ear, demonstrasting a promising possibility to use iron oxide nanoparticles, which are suitable for visualization and characterization at both the light- and electron-microscopic levels.

Array tomography: characterizing FAC-sorted populations of zebrafish immune cells by their 3D ultrastructure.

For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC‐sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high‐throughput ultrastructural imaging of substantial volumes.

In vitro characterization of insulin containing thiomeric microparticles as nasal drug delivery system.

This study focused on a novel two step preparation method for the generation of insulin containing thiomer microparticles. The first step utilized the interpolymer complexation between poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid) (PAA) or poly(acrylic acid)-cysteine (PAA-Cys), respectively, in the presence of insulin. Thereafter lyophilized coprecipitates were micronized via air jet mill. Particles were evaluated regarding size, morphology, insulin release and the effect on ciliary beat frequency of human nasal epithelial cells in vitro. Results displayed mean particle sizes of 2.6±1.6μm and 2.8±1.7μm for PAA/PVP/insulin and PAA-Cys/PVP/insulin microparticles, respectively, in a range where volitional impaction of particles on nasal epithelium takes place. Multi unit dosage forms showed in addition release for the incorporated insulin and nasal safety as to results of ciliary beat frequency studies (CBF). The introduced jet milled microparticles might in conclusion display a safe nasal insulin drug delivery system leading to improved absorption.

On the road to large volumes in LM and SEM: New tools for Array Tomography

1 Cryo EM, Centre for Advanced Materials, Universitat Heidelberg, Heidelberg, Germany 2 HEiKA, Heidelberg Karlsruhe Research Partnership, Heidelberg, Karlsruhe, Germany 3 Institute for Applied Computer Science, Karlsruhe Institute of Technology, Karlsruhe, Germany 4 Carl Zeiss Microscopy GmbH, Oberkochen, Germany 5 RMC Boeckeler, Tucson, Arizona, USA 6 Cryo EM, CellNetworks, BioQuant, Universitatsklinikum Heidelberg, Heidelberg, Germany

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

Assembly and in vitro characterization of thiomeric nanoparticles

Abstract The present study focused on the assembly of an insulin exhibiting, nanoparticulate formulation and the characterization thereof regarding particle size, zeta potential and stability of nanoparticles as well as mucoadhesion indicating, turbidity measurements and drug release studies after particle purification. The preparation was performed in the presence of insulin due to the formation of hydrogen bonds between poly(vinyl pyrrolidone) (PVP) and poly(acrylic acid) (PAA) or its conjugate poly(acrylic acid)-cysteine (PAA-Cys) with a molecular mass of 100 as well as 450 kDa. Stable suspensions, displaying nanoparticles with a mean particle size in the range of 200 nm as well as a negative zeta potential, were achieved with 100 kDa poly(acrylic acid) (PAA100) or poly(acrylic acid)-cysteine (PAA100-Cys). Turbidity measurements displayed a pH dependent interaction of nanoparticulate material and mucin leading to a greater and earlier interference at pH 3.9 compared to pH 7.4. Moreover a 1.2-fold increase of the absorbance of nanoparticle–mucin dispersions compared to mucin control was observed after 3 h. The introduced particulate drug delivery system might in conclusion display a sophisticated vehicle for the non-invasive delivery of insulin and other peptide drugs.

Array Tomography and Beam Deceleration – High-Throughput Imaging with the ZEISS GeminiSEM using Atlas 5 and Beam Deceleration

Utilizing scanning electron microscopy (SEM) to investigate the 3D morphology of cells and tissue has become more and more important in the in the field of biological and medical research. In general two major methods can be distinguished serial block face imaging and Array Tomography [1]. In serial blockface imaging the whole block of resin embedded biological tissue is introduced into the SEM chamber. Then a microtome [2] or focused ion beams [3] are used to cut off a thin top layer of a few nm. An SEM image of the fresh blockface is acquired and the process repeated. Conversely Array Tomography starts by collecting conventional serial section of the resin embedded sample either manually or automatically on larger solid supports which are then introduced into the SEM. Automated image acquisition software is then utilized to record images of hundreds of serial section. Both methods generate hundreds to thousands of sequential images that can be re-aligned to form the 3D volume of the initially sectioned sample.

Correlative large volume imaging across scales

For correlative imaging of large volumes integrated workflows allowing shuttling of samples between different imaging modalities would be ideal. X-ray microscopy (XRM) is one modality, which in principle can bridge the gap between macroscopic and microscopic world. Here we have started to assess how XRM, light microscopy (LM) and electron microscopy (EM) might be integrated to gain comprehensive information about biological samples extended in 3D. In CLEM, correlated LM and EM, fluorescence LM is commonly used to define a certain functional state, which is then put into structural context using EM. That this is also possible for XRM has been shown for single cells grown on TEM grids which were plunge frozen and analyzed by cryo-XRM [1]. For tissue samples such an approach is not possible because samples thicker than 200 micrometers cannot be vitrified. In that case a different workflow is required, starting with a chemical fixation. In LM and XRM samples may be then imaged directly in aqueous medium, XRM being able to yield voxel sizes down to 350 nm. However, if ultrastructural resolution in the range of few nanometers is required XRM needs to be complemented by EM. There are several options to achieve that for large volumes [2] based on SEM imaging such as serial blockface or focussed ion beam scanning electron microscopy (SBFSEM or FIBSEM) or array tomography (AT). SBFSEM is optimal for connectomics where whole brains need to be imaged at high resolution. In other areas of cell and developmental biology the biggest part of the sample surface being imaged may not be interesting for the question being addressed, only a minute part of it may contain the target structure. So the question of how to identify this target is of eminent importance. Here we employ XRM to identify a rare event such as the formation of an immunological synapse (IMS) or a rare structure such as the neuromuscular junction (NMJ) within a large volume. We are using the new solution ZEISS Atlas 5 (Carl Zeiss Microscopy GmbH) which offers a sample centric correlative environment fusing all available 2D and 3D data of the sample from various modalities. It contains modules for automated SEM large area imaging and targeted crossbeam (XB) nanotomography. Based on a large volume XRM dataset it thus permits an efficient approach for nanometer scale analysis of identified buried features of interest using Crossbeam technology. Figure 1 illustrates typical workflows for large volume samples such as muscle tissue or cell pellets. Important is the definition of a reference framework, which is inherent to the samples of interest. This “sample coordinate system” needs to be mapped seamlessly from one imaging technique to the next, which then allows for a fast and accurate navigation of the sample in 3D.