[Ultrastructure of pathologic deposits and cellular inclusions]. / Ultrastruktur pathologischer Ablagerungen und Zellinklusionen.

Intra- and extracellular depositions and inclusions occur in a wide range of diseases with exogenous (e.g. infectious, environmental and toxic) or endogenous (e.g. genetic, inflammatory, neoplastic and degenerative) aetiology. The noxious agent and the pathogenesis influence the organ of manifestation, the subcellular localisation and the ultrastructural appearance of the depositions. Whereas some of the inclusions like pathogens, foreign material (e.g. asbestos) or microvilli have an almost pathognomonic morphology, other inclusions are present in lower amounts also under normal conditions (e.g. lipid vacuoles and glycogen). Therefore, the interpretation of ultrastructural findings makes a correlation with the histological features and clinical constellation necessary. Auxiliary investigations by electron energy loss spectroscopy (EELS) or electron spectroscopic imaging (ESI) provide additional information about the chemical composition of the material and are therefore especially helpful for the identification of foreign substances. This review focuses on a selection of deposits and inclusions relevant to diagnostic pathology.

The influence of chromatic aberration on the dose-limited spatial resolution of transmission electron microscopy.

The effect of chromatic aberration (CC) on the spatial resolution in transmission electron microscopy (TEM) was studied in thick specimens in which the sample becomes the limiting factor in the resolution. The sample influences the energy spread of the electron beam, allows only a limited electron dose, and modulates electron scattering events. The experimental set-up consisted of a thin silicon nitride membrane and a silicon wedge containing gold nanoparticles. The resolution was measured as a function of electron dose and sample thickness for different sample configurations and for different microscopy modalities including regular TEM, energy filtered TEM (EFTEM) and CC-corrected TEM. Comparison with an analytical model aided the understanding of the experimental data applied over varied conditions. The general trend for all microscopy modalities was a transition from a noise-limited resolution at low electron dose to a CC-limited resolution at high-dose in the absence of beam blurring. EFTEM required an accurate energy slit offset and an optimal energy spread to energy-slit width ratio to surpass regular TEM. The key advantage of CC correction appeared to be the best possible resolution for larger sample thickness at low electron dose outperforming EFTEM by about fifty percent. Several hypothetical sample configurations relevant to liquid phase electron microscopy were evaluated as well to demonstrate the capabilities of the analytical model and to determine the most optimal microscopy modality for this type of experiment. The analytical model included an automated optimization of the EFTEM settings and may aid in optimizing the sample-limited resolution for experimental analysis and planning.

Elemental mapping of labelled biological specimens at intermediate energy loss in an energy-filtered TEM acquired using a direct detection device.

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal-to-noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.

Design of dye and superparamagnetic iron oxide nanoparticle loaded lipid nanocapsules with dual detectability in vitro and in vivo.

Lipid nanocapsules are treasured nanoparticulate systems, although they lack detectability in biological environments. To overcome this, we designed LNCs loaded simultaneously with fluorescent dye and superparamagnetic iron oxide nanoparticles (Dual LNCs). The introduction of both labels did not alter nanoparticle characteristics such as size (50 nm), size distribution (polydispersity index < 0.1) or surface modifications, including the effectiveness of targeting ligands. Furthermore, the colloidal stability, particle integrity and biocompatibility of the nanoparticles were not negatively affected by label incorporation. These Dual LNCs are concomitantly visualizable via fluorescence and transmitted light imaging after either the internalization by cells or systemic administration to mice. Importantly, they are detectable in liver sections of mice using transmission electron microscopy without additional enhancement. The iron content of 0.24% (m/m) is sufficiently high for precise quantification of nanoparticle concentrations via inductively coupled plasma optical emission spectroscopy. Dual LNCs are precious tools for the investigation of in vitro and in vivo performances of lipid nanocapsule formulations, since they allow for the use of complementary imaging methods for broad range detectability.

Combining cryo-TEM and energy-filtered TEM for imaging organic core-shell nanoparticles and defining the polymer distribution.

Nanoparticulate systems intended for the use in drug delivery are getting more and more complex. Composite nanoparticles, such as core-shell particles are designed in order to be used for co-delivery of drugs or a modified release profile. Often the structure can only be postulated by the preparation process, such as surface polymerization, but cannot be experimentally determined due to a lack of appropriate analytical methods. Here a core-shell particle system composed of two biodegradable and biocompatible materials, gelatin and PLGA, is developed. In order to reveal the actual polymer distribution, a combination of cryo-transmission electron microscopy and energy-filtered transmission electron microscopy was established. Using the occurrence of specific elements in combination with degradation kinetics induced by the electron beam allows to conclude on the nanoparticles' architecture. Based on these methods and thus, the particle composition, the drug delivery system can be further developed.

EPR imaging of magnetic field effects on radiation dose distributions around millimeter-size air cavities.

New hybrid radiotherapy treatment systems combining an MRI scanner with a source of ionizing radiation are being introduced in the clinic. The strong magnetic fields of MRI considerably affect radiation dose distributions, especially at tissue-air interfaces due to the electron return effect (ERE). Experimental investigation of the ERE within a sub-millimeter thick surface layer is still highly challenging. In the present work, we examine and quantify the magnetic field induced perturbations of dose distributions within a 0.5 mm layer surrounding millimeter-size air cavities by applying electron paramagnetic resonance imaging (EPRI). Air-filled fused quartz tubes (inner diameter 3 or 4 mm) mimic small air cavities and serve as model systems. The tubes were irradiated inside a PMMA phantom by a 6 MV photon beam. The irradiations were performed in the presence or absence of a transverse, magnetic field providing a magnetic field strength of 1.0 Tesla. The spatial distributions of radiation induced paramagnetic defects in the quartz tubes were subsequently determined by applying field-swept echo-detected EPRI and were then converted to relative dose distributions. The transverse magnetic field leads to considerable local dose enhancements and reductions (up to 35%) with respect to the mean dose within the quartz tubes. The experimentally determined dose distributions are in good quantitative agreement with Monte Carlo radiation transport simulations. The results of this work demonstrate the feasibility of field-swept echo-detected EPRI to measure magnetic field induced perturbations of dose distributions within a sub-millimeter thick surface layer at the dosimeter-air interface.

A Histologic Perspective on Electrical and Thermal Burn-Injured Human Skin.

OBJECTIVE: To analyze specific spectroscopic (FT-Raman) and thermal (limiting oxygen index) aspects of skin samples exposed to electrical injury compared with thermal injury. METHODS: An observational case-control study was conducted at the Dr Stanislaw Sakiel Center for Burns Treatment in Siemianowice, Silesia, Poland. A scanning electron microscope was used to diagnose and illustrate the topography of skin samples from electrical and thermal burns and the morphologic effects on damaged versus undamaged skin surfaces. In particular, researchers attempted to detect spectroscopic and thermal changes at the molecular level, namely, specific biomarkers of tissue degeneration and their regeneration under the influence of the applied modifiers (antioxidants and orthosilicic acid solutions). RESULTS: Modification with L-ascorbic acid and hydrogel of orthosilicic acid caused an increase in the intensity of the amide I Raman peaks, whereas modification with sodium ascorbate and orthosilicic acid resulted in the separation of the band protein side chains (1,440-1,448 cm), which is a part of tissue regeneration. The best result was obtained when the skin was treated with 7% orthosilicic acid (limiting oxygen index, 26%). CONCLUSIONS: Antioxidant treatment may be advantageous in minimizing injury in patients with thermal burns but not always in electrical burns.

A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps.

We established a new simple approach to study phosphorus (P) and nitrogen (N) reserves at subcellular level potentially applicable to various types of cells capable of accumulating P- and/or N-rich inclusions. Here, we report on using this approach for locating and assessing the abundance of the P and N reserves in microalgal and cyanobacterial cells. The approach includes separation of the signal from P- or N-rich structures from noise on the energy-filtered transmission electron microscopy (EFTEM) P- or N-maps. The separation includes (i) relative entropy estimation for each pixel of the map, (ii) binary thresholding of the map, and (iii) segmenting the image to assess the inclusion relative area and localization in the cell section. The separation is based on comparing the a posteriori probability that a pixel of the map contains information about the sample vs. Gaussian a priori probability that the pixel contains noise. The difference is expressed as relative entropy value for the pixel; positive values are characteristic of the pixels containing the payload information about the sample. This is the first known method for quantification and locating at a subcellular level P-rich and N-rich inclusions including tiny (< 180 nm) structures. We demonstrated the applicability of the proposed method both to the cells of eukaryotic green microalgae and cyanobacteria. Using the new method, we elucidated the heterogeneity of the studied cells in accumulation of P and N reserves across different species. The proposed approach will be handy for any cytological and microbiological study requiring a comparative assessment of subcellular distribution of cyanophycin, polyphosphates or other type of P- or N-rich inclusions. An added value is the potential of this approach for automation of the data processing and evaluation enabling an unprecedented increase of the EFTEM analysis throughput.

LED for hyperspectral imaging - a new selection method.

Hyperspectral imaging (HSI) has become a sophisticated technique in modern applications such as food analyses, recycling technology, medicine, pharmacy and forensic science. It allows one to analyse both spatial and spectral information from an object. But hyperspectral cameras are still expensive due to their extended wavelength range. The development of new light-emitting diodes (LED) in the recent past enables another approach to HSI using a monochrome camera in combination with a LED-based illumination. However, such a system has a lower spectral resolution. Additionally, the growing supply of LED on the market complicates the selection of LED. In this paper, we propose a new time efficient selection method for the design process of an illumination. It chooses an optimised LED combination from an existing database to match a predefined spectral power distribution. Therefore, an algorithm is used to evaluate various LED combinations. Furthermore, the method considers the spectral behaviour of each LED in dependence of forward current and temperature of the solder point. Our method has already shown promise during the selection process for even spectral distributions which is demonstrated in the study. Additionally, we will show its potential for HSI illuminations.

Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy.

Bone is a highly organized tissue in which each structural level influences the macroscopic and microscopic mechanical behavior. In particular, the quantity, quality, and distribution of the different bone components, i.e. collagen matrix and hydroxyapatite crystals, are associated with bone strength or fragility. Common spectroscopic techniques used to assess bone composition have resolutions limited to the micrometer range. In this study, our aims were two-fold: i) to develop and validate the AFM-IR methodology for skeletal tissues and ii) to apply the methodology to sheep cancellous bone with the objective to obtain novel findings on the composition and structure of trabecular packets.To develop the methodology, we assessed spatial and temporal reproducibility using a known homogeneous material (polymethylmethacrylate, PMMA). We verified that the major peak positions were similar and not shifted when compared to traditional Fourier Transform Infrared imaging (FTIRI). When AFM-IR was applied to sheep cancellous bone, the mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased as a function of tissue maturity. The resolution of the technique enabled visualization of different stages of the bone maturation process, particularly newly-formed osteoid prior to mineralization. We also observed alternating patterns of IR parameters in line and imaging measurements, suggesting the apposition of layers of alternating structure and / or composition that were not visible with traditional spectroscopic methods. In conclusion, nanoscale IR spectroscopy demonstrates novel compositional and structural changes within trabecular packets in cancellous bone. Based on these results, AFM-IR is a valuable tool to investigate cancellous bone at the nanoscale and, more generally, to analyze small dynamic areas that are invisible to traditional spectroscopic methods.

Colocalization of Aluminum and Iron in Nuclei of Nerve Cells in Brains of Patients with Alzheimer's Disease.

Increasing evidence indicates that metal-induced oxidative stress plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). Recently, the presence of 8-hydroxydeoxyguanosine, a biomarker of oxidative DNA damage, was demonstrated in nuclear DNA (nDNA) in the AD brain. Iron (Fe) is a pro-oxidant metal capable of generating hydroxyl radicals that can oxidize DNA, and aluminum (Al) has been reported to facilitate Fe-mediated oxidation. In the present study, we examined the elements contained in the nuclei of nerve cells in AD brains using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS). Our results demonstrated that Al and Fe were colocalized in the nuclei of nerve cells in the AD brain. Within the nuclei, the highest levels of both Al and Fe were measured in the nucleolus. The SEM-EDS analysis also revealed the colocalization of Al and Fe in the heterochromatin and euchromatin in neuronal nuclei in the AD brain. Notably, the levels of Al and Fe in the nucleus of nerve cells in the AD brain were markedly higher than those in age-matched control brains. We hypothesize that the colocalization of Al and Fe in the nucleus of nerve cells might induce oxidative damage to nDNA and concurrently inhibit the repair of oxidatively damaged nDNA. An imbalance caused by the increase in DNA damage and the decrease in DNA repair activities might lead to the accumulation of unrepaired damaged DNA, eventually causing neurodegeneration and the development of AD.

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography.

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

Atomic resolution elemental mapping using energy-filtered imaging scanning transmission electron microscopy with chromatic aberration correction.

This paper addresses a novel approach to atomic resolution elemental mapping, demonstrating a method that produces elemental maps with a similar resolution to the established method of electron energy-loss spectroscopy in scanning transmission electron microscopy. Dubbed energy-filtered imaging scanning transmission electron microscopy (EFISTEM) this mode of imaging is, by the quantum mechanical principle of reciprocity, equivalent to tilting the probe in energy-filtered transmission electron microscopy (EFTEM) through a cone and incoherently averaging the results. In this paper we present a proof-of-principle EFISTEM experimental study on strontium titanate. The present approach, made possible by chromatic aberration correction, has the advantage that it provides elemental maps which are immune to spatial incoherence in the electron source, coherent aberrations in the probe-forming lens and probe jitter. The veracity of the experiment is supported by quantum mechanical image simulations, which provide an insight into the image-forming process. Elemental maps obtained in EFTEM suffer from the effect known as preservation of elastic contrast, which, for example, can lead to a given atomic species appearing to be in atomic columns where it is not to be found. EFISTEM very substantially reduces the preservation of elastic contrast and yields images which show stability of contrast with changing thickness. The experimental application is demonstrated in a proof-of-principle study on strontium titanate.

Application of EELS and EFTEM to the life sciences enabled by the contributions of Ondrej Krivanek.

The pioneering contributions of Ondrej Krivanek to the development of electron energy loss spectrometers, energy filters, and detectors for transmission and scanning transmission electron microscopes have provided researchers with indispensible tools across a wide range of disciplines in the physical sciences, ranging from condensed matter physics, to chemistry, mineralogy, materials science, and nanotechnology. In addition, the same instrumentation has extended its reach into the life sciences, and it is this aspect of Ondrej Krivanek's influential contributions that will be surveyed here, together with some personal recollections. Traditionally, electron microscopy has given a purely morphological view of the biological structures that compose cells and tissues. However, the availability of high-performance electron energy loss spectrometers and energy filters offers complementary information about the elemental and chemical composition at the subcellular scale. Such information has proven to be valuable for applications in cell and structural biology, microbiology, histology, pathology, and more generally in the biomedical sciences.

Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels.IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

Nitroxide Spin-Labelling and Its Role in Elucidating Cuproprotein Structure and Function.

Copper is one of the most abundant biological metals, and its chemical properties mean that organisms need sophisticated and multilayer mechanisms in place to maintain homoeostasis and avoid deleterious effects. Studying copper proteins requires multiple techniques, but electron paramagnetic resonance (EPR) plays a key role in understanding Cu(II) sites in proteins. When spin-labels such as aminoxyl radicals (commonly referred to as nitroxides) are introduced, then EPR becomes a powerful technique to monitor not only the coordination environment, but also to obtain structural information that is often not readily available from other techniques. This information can contribute to explaining how cuproproteins fold and misfold. The theory and practice of EPR can be daunting to the non-expert; therefore, in this mini review, we explore how nitroxide spin-labelling can be used to help the inorganic biochemist gain greater understanding of cuproprotein structure and function in vitro and how EPR imaging may help improve understanding of copper homoeostasis in vivo.

Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.

The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. IMPORTANCE: How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane.