The influence of size and surface chemistry on the bioavailability, tissue distribution and toxicity of gold nanoparticles in zebrafish (Danio rerio).

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10-100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40-80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

High activity and high functional connectivity are mutually exclusive in resting state zebrafish and human brains.

BACKGROUND: The structural connectivity of neurons in the brain allows active neurons to impact the physiology of target neuron types with which they are functionally connected. While the structural connectome is at the basis of functional connectome, it is the functional connectivity measured through correlations between time series of individual neurophysiological events that underlies behavioral and mental states. However, in light of the diverse neuronal cell types populating the brain and their unique connectivity properties, both neuronal activity and functional connectivity are heterogeneous across the brain, and the nature of their relationship is not clear. Here, we employ brain-wide calcium imaging at cellular resolution in larval zebrafish to understand the principles of resting state functional connectivity. RESULTS: We recorded the spontaneous activity of >12,000 neurons in the awake resting state forebrain. By classifying their activity (i.e., variances of ΔF/F across time) and functional connectivity into three levels (high, medium, low), we find that highly active neurons have low functional connections and highly connected neurons are of low activity. This finding holds true when neuronal activity and functional connectivity data are classified into five instead of three levels, and in whole brain spontaneous activity datasets. Moreover, such activity-connectivity relationship is not observed in randomly shuffled, noise-added, or simulated datasets, suggesting that it reflects an intrinsic brain network property. Intriguingly, deploying the same analytical tools on functional magnetic resonance imaging (fMRI) data from the resting state human brain, we uncover a similar relationship between activity (signal variance over time) and functional connectivity, that is, regions of high activity are non-overlapping with those of high connectivity. CONCLUSIONS: We found a mutually exclusive relationship between high activity (signal variance over time) and high functional connectivity of neurons in zebrafish and human brains. These findings reveal a previously unknown and evolutionarily conserved brain organizational principle, which has implications for understanding disease states and designing artificial neuronal networks.

Applications and Advances of Multicellular Tumor Spheroids: Challenges in Their Development and Analysis.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

Multi-sample SPIM image acquisition, processing and analysis of vascular growth in zebrafish.

To quantitatively understand biological processes that occur over many hours or days, it is desirable to image multiple samples simultaneously, and automatically process and analyse the resulting datasets. Here, we present a complete multi-sample preparation, imaging, processing and analysis workflow to determine the development of the vascular volume in zebrafish. Up to five live embryos were mounted and imaged simultaneously over several days using selective plane illumination microscopy (SPIM). The resulting large imagery dataset of several terabytes was processed in an automated manner on a high-performance computer cluster and segmented using a novel segmentation approach that uses images of red blood cells as training data. This analysis yielded a precise quantification of growth characteristics of the whole vascular network, head vasculature and tail vasculature over development. Our multi-sample platform demonstrates effective upgrades to conventional single-sample imaging platforms and paves the way for diverse quantitative long-term imaging studies.

Gibbs point field model quantifies disorder in microvasculature of U87-glioblastoma.

Microvascular proliferation in glioblastoma multiforme is a biological key mechanism to facilitate tumor growth and infiltration and a main target for treatment interventions. The vascular architecture can be obtained by Single Plane Illumination Microscopy (SPIM) to evaluate vascular heterogeneity in tumorous tissue. We make use of the Gibbs point field model to quantify the order of regularity in capillary distributions found in the U87 glioblastoma model in a murine model and to compare tumorous and healthy brain tissue. A single model parameter Γ was assigned that is linked to tissue-specific vascular topology through Monte-Carlo simulations. Distributions of the model parameter Γ differ significantly between glioblastoma tissue with mean 〈ΓG〉=2.1±0.4, as compared to healthy brain tissue with mean 〈ΓH〉=4.9±0.4, suggesting that the average Γ-value allows for tissue differentiation. These results may be used for diagnostic magnetic resonance imaging, where it has been shown recently that Γ is linked to tissue-inherent relaxation parameters.

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm.

Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are demonstrated. Images of beads prove a near-isotropic lateral resolution of sub-100 nm. Images of yeast endoplasmic reticulum show that coherent structured illumination (csi) LSFM performs with physiologically relevant specimens.

Seeing is believing - multi-scale spatio-temporal imaging towards in vivo cell biology.

Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, super-resolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease.

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis.

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen-age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior-posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis-diffusion-degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen-age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.

Calcium transport into the cells of the sea urchin larva in relation to spicule formation.

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.

Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development.

Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy. Multimodal imaging techniques are now needed to put these distinct structures back into the context of the surrounding tissue. We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity. We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.

Investigating the RAS can be a fishy business: interdisciplinary opportunities using Zebrafish.

The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for in vivo imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides.

3D print customized sample holders for live light sheet microscopy.

A major hurdle to the widespread application of light sheet microscopy is the lack of versatile and non-intrusive sample holders that are adaptable to a variety of biological samples for live imaging. To overcome this limitation, we present herein the application of 3D printing to the fabrication of a fully customizable casting kit. 3D printing enables facile preparation of hydrogel sample holders adaptable to any shape and number of specimen. As an example, we present the use of this device to produce a four-sample holder adapted to parallel live monitoring of multicellular tumor spheroid growth. To share our solution with the light sheet microscopy community, all files necessary to produce or customize sample holders are freely available online.

Sample holder for axial rotation of specimens in 3D microscopy.

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

Single-molecule super-resolution light-sheet microscopy.

Single-molecule super-resolution imaging is a new promising tool for investigation of sub-cellular structures. Concurrently, light-sheet microscopy, also known as selective plane illumination microscopy (SPIM), has gained rapid favor with the imaging community in developmental biology due to its fast speed, high contrast, deep penetration, and low phototoxicity. While nearly a dozen reviews thoroughly describe the development of light-sheet microscopy and its technological breakthroughs with a main focus on improving the 3D imaging speed of fish embryos, central nervous system, and other tissues, few have addressed the potential of combining light-sheet microscopy and localization-based super-resolution imaging to achieve sub-diffraction-limited resolution. Adapting light-sheet illumination for single-molecule imaging presents unique challenges for instrumentation and reconstruction algorithms. In this Minireview, we provide an overview of the recent developments that address these challenges. We compare different approaches in super-resolution and light-sheet imaging, address advantages and limitations in each approach, and outline future directions of this emerging field.

A versatile sample holder for single plane illumination microscopy.

Single Plane Illumination Microscopy is an emerging and powerful technology for live imaging of whole living organisms. However, sample handling that relies on specimen embedding in agarose or gel is often a key limitation, especially for time-lapse monitoring. To address this issue, we developed a new concept for a holder device allowing us to prepare a sample container made of hydrogel. The production process of this holder is based on 3D printing of both a frame and casting devices. The simplicity of production and the advantages of this versatile new sample holder are shown with time-lapse recording of multicellular tumour spheroid growth. More importantly, we also show that cell division is not impaired in contrast to what is observed with gel embedding. The benefit of this new holder for other sample types, applications and experiments remains to be evaluated, but this innovative concept of fully customizable sample holder preparation potentially represents a major step forward to facilitate the large diffusion of single plane illumination microscopy technology.

Advanced Computational Methods to Evaluate Vascular Heterogeneity in Tumor Tissue Based on Single Plane Illumination Microscopy.

During tumor growth, the complex composition of vasculature is prone to dynamic changes due to mechanic and biochemical challenges. Perivascular invasion of tumor cells to co-opt existing vasculature, but also formation of de-novo vasculature and other effects on the vascular network, may lead to altered geometric vessel properties as well as changes in vascular network topology, which is defined by vascular multifurcations and connections between vessel segments. The intricate organization and heterogeneity of the vascular network can be analyzed with advanced computational methods to uncover vascular network signatures that may allow differentiating between pathological and physiological vessel regions. Herein, we present a protocol to evaluate vascular heterogeneity in whole vascular networks, using morphological and topological measures. The protocol was developed for single plane illumination microscopy images of mice brain vasculature but can be applied to any vascular network.

A 3D Analysis of Cleared Human Melanoma.

Cutaneous melanoma is one of the most aggressive and deadliest cancers in human beings due to its invasiveness and other factors. Histopathological analysis is crucial for a proper diagnosis. Optical tissue clearing is a novel field that allows 3D image acquisition of large-scale biological tissues. Optical clearing and immunolabeling for 3D fluorescence imaging has yet to be extensively applied to melanoma. In the present manuscript, we establish, for the first time, an optical clearing and immunostaining procedure for human melanoma and human cell line-derived melanoma xenograft models using the CUBIC (clear, unobstructed brain imaging cocktails) technique. We have successfully cleared the samples and achieved 3D volumetric visualization of the tumor microenvironment, vasculature, and cell populations.

Research of High-Purity Lanthanum Prepared by Zone Refining.

In this paper, the purification of lanthanum was studied by using zone-refining technology. The equilibrium distribution coefficient of impurities was calculated using a liquidus slope method to reveal impurities' distribution properties. Meanwhile, the analysis of impurities' concentration distribution for Fe and Si has been investigated based on the SPIM model. The calculated findings based on SPIM were found to be in good agreement with the experimental results. In addition, the influence of the zone-refining rate and the number of passes on the purification of lanthanum were studied. It was found that after ten times of zone refining with a zone-refining rate of 5 mm/min, the contents of Fe and Si impurities in metal decreased to 4 and 2 ppm, respectively.

Design of a versatile microfluidic device for imaging precision-cut-tissue slices.

Precision-cut-tissues (PCTs), which preserve many aspects of a tissue's microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice. We report here on the design of a versatile microfluidic device capable of holding mouse or human pancreas PCTs for 3D fluorescence imaging using confocal and selective plane illumination microscopy (SPIM). Our design positions PCTs within a 5 × 5 mm × 140µm deep chamber fitted with 150µm tall channels to facilitate media exchange. Shear stress in the device is localized to small regions on the surface of the tissue and can be easily controlled. This design allows for media exchange at flowrates ∼10-fold lower than those required for conventional chambers. Finally, this design allows for imaging the same immunofluorescently labeled PCT with high resolution on a confocal and with large field of view on a SPIM, without adversely affecting image quality.

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal "snapshots" of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination, 3D SMLM, and protein labeling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analyzing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely, or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.