Eric W. Roth

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR–pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice

Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle–scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.

Imaging and elemental mapping of biological specimens with a dual-EDS dedicated scanning transmission electron microscope

A dedicated analytical scanning transmission electron microscope (STEM) with dual energy dispersive spectroscopy (EDS) detectors has been designed for complementary high performance imaging as well as high sensitivity elemental analysis and mapping of biological structures. The performance of this new design, based on a Hitachi HD-2300A model, was evaluated using a variety of biological specimens. With three imaging detectors, both the surface and internal structure of cells can be examined simultaneously. The whole-cell elemental mapping, especially of heavier metal species that have low cross-section for electron energy loss spectroscopy (EELS), can be faithfully obtained. Optimization of STEM imaging conditions is applied to thick sections as well as thin sections of biological cells under low-dose conditions at room and cryogenic temperatures. Such multimodal capabilities applied to soft/biological structures usher a new era for analytical studies in biological systems.

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice.

The effects of chemical fixation on the cellular nanostructure

ABSTRACT Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo‐fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever‐finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label‐free live cell spectroscopic‐imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo‐fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods. HIGHLIGHTSPWS was employed to monitor fixation process for the same cells in vitro.Dramatic changes in cellular nanostructure were observed after fixation.However, the population difference of chromatin structure remains after fixation.

Suppressing Electron Exposure Artifacts: An Electron Scanning Paradigm with Bayesian Machine Learning.

Electron microscopy of biological, polymeric, and other beam-sensitive structures is often hampered by deleterious electron beam interactions. In fact, imaging of such beam-sensitive materials is limited by the allowable radiation dosage rather that capabilities of the microscope itself, which has been compounded by the availability of high brightness electron sources. Reducing dwell times to overcome dose-related artifacts, such as radiolysis and electrostatic charging, is challenging due to the inherently low contrast in imaging of many such materials. These challenges are particularly exacerbated during dynamic time-resolved, fluidic cell imaging, or three-dimensional tomographic reconstruction-all of which undergo additional dosage. Thus, there is a pressing need for the development of techniques to produce high-quality images at ever lower electron doses. In this contribution, we demonstrate direct dose reduction and suppression of beam-induced artifacts through under-sampling pixels, by as much as 80% reduction in dosage, using a commercial scanning electron microscope with an electrostatic beam blanker and a dictionary learning in-painting algorithm. This allows for multiple sparse recoverable images to be acquired at the cost of one fully sampled image. We believe this approach may open new ways to conduct imaging, which otherwise require compromising beam current and/or exposure conditions.

High speed/low dose analytical electron microscopy with dynamic sampling

Technological advances in electron microscopy, particularly improved detectors and aberration correctors, have led to higher throughput and less invasive imaging of materials and biological structures by enhancing signal-to-noise ratios at lower electron exposures. Analytical methods, such as electron energy loss spectroscopy (EELS) and energy dispersive x-ray spectrometry (EDS), have also benefitted and offer a rich set of local elemental and bonding information with nano-or atomic resolution. However, spatially resolved spectrum imaging with EELS and EDS continue to be difficult to scale due to limited detector collection angles or high signal background, requiring hours or even days for full maps. We present the principle and application of a Multi-Objective Autonomous Dynamic Sampling (MOADS) method which can accelerate spectrum mapping in EELS or EDS by over an order of magnitude. Initial guesses about the true spectrum images are constructed as measurements are collected, which allows the prediction of points which contribute information/contrast. In this fashion, an intelligently selected and reduced set of points which best approximate the true spectrum image are autonomously collected on-the-fly to save considerable time and/or radiative area dose. We implemented MOADS as a software add-on to arbitrary commercial Scanning Transmission Electron Microscopes (STEMs) equipped with a Gatan Digital Micrograph (DM, Gatan ©) interface. We demonstrate the efficacy of our proposed method on several prototypical analytical specimens, as well as dose sensitive materials. It is expected that MOADS and similar supervised dynamic sampling approaches may open the exploration of large area analytical maps or the imaging of beam reactive materials not previously thought feasible.

Optothermally Reversible Carbon Nanotube–DNA Supramolecular Hybrid Hydrogels

Supramolecular hydrogels (SMHs) are three-dimensional constructs wherein the majority of the volume is occupied by water. Since the bonding forces between the components of SMHs are noncovalent, SMH properties are often tunable, stimuli-responsive, and reversible, which enables applications including triggered drug release, sensing, and tissue engineering. Meanwhile, single-walled carbon nanotubes (SWCNTs) possess superlative electrical and thermal conductivities, high mechanical strength, and strong optical absorption at near-infrared wavelengths that have the potential to add unique functionality to SMHs. However, SWCNT-based SMHs have thus far not realized the potential of the optical properties of SWCNTs to enable reversible response to near-infrared irradiation. Here, we present a novel SMH architecture comprised solely of DNA and SWCNTs, wherein noncovalent interactions provide structural integrity without compromising the intrinsic properties of SWCNTs. The mechanical properties of these SMHs are readily tuned by varying the relative concentrations of DNA and SWCNTs, which varies the cross-linking density as shown by molecular dynamics simulations. Moreover, the SMH gelation transition is fully reversible and can be triggered by a change in temperature or near-infrared irradiation. This work explores a new regime for SMHs with potential utility for a range of applications including sensors, actuators, responsive substrates, and 3D printing.

Spectroscopic and Microscopic Evidence of Biomediated HgS Species Formation from Hg(II)–Cysteine Complexes: Implications for Hg(II) Bioavailability

We investigated the chemistry of Hg(II) during exposure of exponentially growing bacteria ( Escherichia coli, Bacillus subtilis, and Geobacter sulfurreducens) to 50 nM, 500 nM, and 5 μM total Hg(II) with and without added cysteine. With X-ray absorption spectroscopy, we provide direct evidence of the formation of cell-associated HgS for all tested bacteria. The addition of cysteine (100-1000 μM) promotes HgS formation (>70% of total cell-associated Hg(II)) as a result of the biodegradation of added cysteine to sulfide. Cell-associated HgS species are also detected when cysteine is not added as a sulfide source. Two phases of HgS, cinnabar (α-HgS) and metacinnabar (β-HgS), form depending on the total concentration of Hg(II) and sulfide in the exposure medium. However, α-HgS exclusively forms in assays that contain an excess of cysteine. Scanning transmission electron microscopy images reveal that nanoparticulate HgS(s) is primarily located at the cell surface/extracellular matrix of Gram-negative E. coli and G. sulfurreducens and in the cytoplasm/cell membrane of Gram-positive B. subtilis. Intracellular Hg(II) was detected even when the predominant cell-associated species was HgS. This study shows that HgS species can form from exogenous thiol-containing ligands and endogenous sulfide in Hg(II) biouptake assays under nondissimilatory sulfate reducing conditions, providing new considerations for the interpretation of Hg(II) biouptake results.

Nanoscale chromatin structure characterization for optical applications: a transmission electron microscopy study (Conference Presentation)

Structural and biological origins of light scattering in cells and tissue are still poorly understood. We demonstrate how this problem might be addressed through the use of transmission electron microscopy (TEM). For biological samples, TEM image intensity is proportional to mass-density, and thus proportional to refractive index (RI). By calculating the autocorrelation function (ACF) of TEM image intensity of a thin-section of cells, we essentially maintain the nanoscale ACF of the 3D cellular RI distribution, given that the RI distribution is statistically isotropic. Using this nanoscale 3D RI ACF, we can simulate light scattering through biological samples, and thus guiding many optical techniques to quantify specific structures. In this work, we chose to use Partial Wave Spectroscopy (PWS) microscopy as a one of the nanoscale-sensitive optical techniques. Hela cells were prepared using standard protocol to preserve nanoscale ultrastructure, and a 50-nm slice was sectioned for TEM imaging at 6 nm resolution. The ACF was calculated for chromatin, and the PWS mean sigma was calculated by summing over the power spectral density in the visible light frequency of a random medium generated to match the ACF. A 1-µm slice adjacent to the 50-nm slice was sectioned for PWS measurement to guarantee identical chromatin structure. For 33 cells, we compared the calculated PWS mean sigma from TEM and the value measured directly, and obtained a strong correlation of 0.69. This example indicates the great potential of using TEM measured RI distribution to better understand the quantification of cellular nanostructure by optical methods.