Stacked Denoising Autoencoders and Transfer Learning for Immunogold Particles Detection and Recognition

In this paper we present a system for the detection of immunogold particles and a Transfer Learning (TL) framework for the recognition of these immunogold particles. Immunogold particles are part of a high-magnification method for the selective localization of biological molecules at the subcellular level only visible through Electron Microscopy. The number of immunogold particles in the cell walls allows the assessment of the differences in their compositions providing a tool to analise the quality of different plants. For its quantization one requires a laborious manual labeling (or annotation) of images containing hundreds of particles. The system that is proposed in this paper can leverage significantly the burden of this manual task. For particle detection we use a LoG filter coupled with a SDA. In order to improve the recognition, we also study the applicability of TL settings for immunogold recognition. TL reuses the learning model of a source problem on other datasets (target problems) containing particles of different sizes. The proposed system was developed to solve a particular problem on maize cells, namely to determine the composition of cell wall ingrowths in endosperm transfer cells. This novel dataset as well as the code for reproducing our experiments is made publicly available. We determined that the LoG detector alone attained more than 84\% of accuracy with the F-measure. Developing immunogold recognition with TL also provided superior performance when compared with the baseline models augmenting the accuracy rates by 10\%

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin

Background: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Methods: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Results: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. Conclusions: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns

Cerebral endothelial cell derived small extracellular vesicles improve cognitive function in aged diabetic rats

Small extracellular vesicles (sEVs) mediate cell-cell communication by transferring their cargo biological materials into recipient cells. Diabetes mellitus (DM) induces cerebral vascular dysfunction and neurogenesis impairment, which are associated with cognitive decline and an increased risk of developing dementia. Whether the sEVs are involved in DM-induced cerebral vascular disease, is unknown. Therefore, we studied sEVs derived from cerebral endothelial cells (CEC-sEVs) of aged DM rats (DM-CEC-sEVs) and found that DM-CEC-sEVs robustly inhibited neural stem cell (NSC) generation of new neuroblasts and damaged cerebral endothelial function. Treatment of aged DM-rats with CEC-sEVs derived from adult healthy normal rats (N-CEC-sEVs) ameliorated cognitive deficits and improved cerebral vascular function and enhanced neurogenesis. Intravenously administered N-CEC-sEVs crossed the blood brain barrier and were internalized by neural stem cells in the neurogenic region, which were associated with augmentation of miR-1 and -146a and reduction of myeloid differentiation primary response gene 88 and thrombospondin 1 proteins. In addition, uptake of N-CEC-sEVs by the recipient cells was mediated by clathrin and caveolin dependent endocytosis signaling pathways. The present study provides ex vivo and in vivo evidence that DM-CEC-sEVs induce cerebral vascular dysfunction and neurogenesis impairment and that N-CEC-sEVs have a therapeutic effect on improvement of cognitive function by ameliorating dysfunction of cerebral vessels and increasing neurogenesis in aged DM rats, respectively

In situ cryo-correlative light and electron tomography of influenza A virus entry and its inhibition by IFITM3

The innate immune system is the first wall of defense against many infectious pathogens, such as viruses or bacteria. The antiviral interferon-induced transmembrane protein 3 (IFITM3) is one of the key players against enveloped viruses like the influenza A virus. IFITM3 is localized in the endosomal-lysosomal system and is known to prevent viral cytoplasmic entry. Different hypotheses on the mode of action of IFITM3 were proposed, but the underlying molecular mechanism still needs to be fully understood. Here, I am using a combination of cryo-light microscopy and in situ cryo-electron tomography to study the antiviral function of IFITM3 within the natural cellular environment in the context of an influenza A virus infection. To visualize the antiviral actions of IFITM3, I established a novel cryo-correlative light and electron microscopy method. This novel approach allowed me to localize trapped influenza A virus particles in the endosomal-lysosomal system of an IFITM3-overexpressing human epithelial lung cell line A549, which allowed me to study them by cryo-electron tomography. Structural analysis of IFITM3-positive multivesicular bodies revealed that IFITM3 does not alter the ultrastructural morphology of the endosomal-lysosomal system and does not modulate the number of intraluminal vesicles (ILVs). These results contradict the ’fusion decoy hypothesis,’ which suggests that an increased number of ILVs in the late endosomal lumen could redirect viral membrane fusion from the limiting late endosomal membrane to fusion with ILVs. High-resolution in situ cryo-electron tomography of influenza A virus particles within late endosomes revealed that IFITM3 traps influenza A virus particles in a hemifusion state at the limiting late endosomal membrane and ILVs. These findings support the previously formulated ’hemifusion stabilization’ hypothesis as they are the first direct proof of IFITM3-mediated hemifusion stabilization within the natural cellular environment. Furthermore, ultrastructural characterization of the hemifusion sites revealed the post-fusion form of the viral fusion protein hemagglutinin (HA). Thus, IFITM3 does not inhibit low-pH triggered HA conformational changes, indicating that IFITM3 inhibits membrane fusion indirectly by modulating the membrane properties of the late endosomal-lysosomal system and thus stabilizing hemifusion

Chip-scale bioassays based on surface-enhanced Raman scattering: fundamentals and applications

This work explores the development and application of chip-scale bioassays based on surface-enhanced Raman scattering (SERS) for high throughput and high sensitivity analysis of biomolecules;The size effect of gold nanoparticles on the intensity of SERS is first presented. A sandwich immunoassay was performed using Raman-labeled immunogold nanoparticles with various sizes. The SERS responses were correlated to particle densities, which were obtained by atomic force microscopy (AFM). The response of individual particles was also investigated using Raman-microscope and an array of gold islands on a silicon substrate. The location and the size of individual particles were mapped using AFM;The next study describes a low-level detection of Escherichia coli O157:H7 and simulants of biological warfare agents in a sandwich immunoassay format using SERS labels, which have been termed Extrinsic Raman labels (ERLs). A new ERL scheme based on a mixed monolayer is also introduced. The mixed monolayer ERLs were created by covering the gold nanoparticles with a mixture of two thiolates, one thiolate for covalently binding antibody to the particle and the other thiolate for producing a strong Raman signal;An assay platform based on mixed self-assembled monolayers (SAMs) on gold is then presented. The mixed SAMs were prepared from dithiobis(succinimidyl undecanoate) (DSU) to covalently bind antibodies on gold substrate and oligo(ethylene glycol)-terminated thiol to prevent nonspecific adsorption of antibodies. After the mixed SAMs surfaces, formed from various mole fraction of DSU were incubated with antibodies, AFM was used to image individual antibodies on the surface;The final study presents a collaborative work on the single molecule adsorption of YOYO-I labeled lambda-DNA at compositionally patterned SAMs using total internal reflection fluorescence microscopy. The role of solution pH, lambda-DNA concentration, and domain size was investigated. This work also revealed the potential importance of structural defects

Mitochondrial Fission After Traumatic Brain Injury

Mitochondrial dysfunction is a central feature in the pathophysiology of Traumatic Brain Injury (TBI). Loss of mitochondrial function disrupts normal cellular processes in the brain, as well as impedes the ability for repair and recovery, creating a vicious cycle that perpetuates damage after injury. To maintain metabolic homeostasis and cellular health, mitochondria constantly undergo regulated processes of fusion and fission and functionally adapt to changes in the cellular environment. An imbalance of these processes can disrupt the ability for mitochondria to functionally meet the metabolic needs of the cell, therefore resulting in mitochondrial damage and eventual cell death. Excessive fission, in particular, has been identified as a key pathological event in neuronal damage and death in many neurodegenerative disease models. Specifically, dysregulation of the primary protein regulator of mitochondrial fission, Dynamin-related Protein 1 (Drp1), has been implicated as an underlying mechanism associated with excessive fission and neurodegeneration; however, whether dysregulation of Drp1 and excessive fission occur after TBI and contribute to neuropathological outcome is not well known. The studies described in this dissertation investigate the following hypothesis: TBI causes dysregulation of Drp1 and increases mitochondrial fission in the hippocampus, and inhibiting Drp1 will reduce mitochondrial dysfunction, reduce neuronal damage, and improve cognitive function after injury. Results from these studies revealed four key findings: 1) Experimental TBI increases Drp1 association with mitochondria, and 2) causes acute changes in Drp1-mediated mitochondrial morphology that persists post-injury, indicating increased mitochondrial fission acutely after injury. Additionally, 3) post-injury treatment with a pharmacological inhibitor of Drp1, Mdivi-1, improved survival of newly born neurons in the injured hippocampus, and 4) improved hippocampal-dependent cognitive function after experimental TBI. Taken together, results from these studies reveal that TBI causes excessive Drp1-mediated mitochondrial fission and that this pathological fission state may play a key role in hippocampal neuronal death and cognitive deficits after TBI. Furthermore, these findings indicate inhibition of Drp1 and mitochondrial fission as a potential therapeutic strategy to improve neuronal recovery and cognitive function after injury

Development of a Novel Super Resolution Microscopy Technique using an Electron Beam for High Resolution Imaging

The requirement for spatial resolution that surpasses the limitations of classical light microscopy has proceeded along two paths; super-resolution light microscopy and electron microscopy. Optical microscopy offers multicolour flexibility for use in live cell imaging. Conversely, electron microscopes require a vacuum, and are essentially monochromatic; biological materials require dehydration and laborious treatments. Correlative light and electron microscopy endeavours to combine the two imaging approaches to maximise spatial resolution and correlate these images with live cell dynamics. The JEOL ClairScope atmospheric scanning electron microscope (ASEM) enables wide-field fluorescence microscopy on living cells, and can then obtain electron microscope images on the same sample in a hydrated environment. In many cases this would use conventional heavy metal stains; this project aimed to use the instrument for investigations of specific biological interactions, using immunogold. The labelling protocols were verified using the model system of actin in cells. The achievable resolution of the ClairScope was determined to be about 20 nm. Immunogold labelling was used to investigate the recycling dynamics of vesicles in neuroblastoma cells. The use of fluid phase markers such as FM dyes were used in attempts to correlate fluorescence and ASEM, although it was discovered that in an aqueous environment these markers were not suitable. The immunogold labelling system was further employed to investigate of the previously reported association of the protein kinase A (PKA) subunit RIα. Puncta of this protein were observed to form during differentiation of neuroblastoma cells following treatment with cAMP. Various techniques in fluorescence microscopy were employed in the study of RIα localisation. Photobleaching revealed that RIα puncta freely exchanged with cytoplasmic molecules. Further work is required to shed light on the signalling mechanism of neuroblastoma differentiatio

Imaging Disease-related Protein Aggregates Inside Human Cells Using a Selenium Label

The aberrant folding and subsequent aggregation of proteins into insoluble plaques known as amyloid fibrils is associated with a number of diseases, including Alzheimer’s and Parkinson’s diseases. The exact role that these aggregates play in the disease mechanisms is not yet well understood, in part due to the difficulties that arise when attempting to visualise the interactions between the carbon-rich protein aggregates and the carbon-rich cells due to a lack of contrast. Traditional strategies to overcome this lack of contrast have involved the use of stains or tags that potentially can be either unreliable or intrusive. In this work we have taken a fragment of the Alzheimer’s-related Aβ peptide and replaced the naturally occurring sulfur that is present in the methionine amino acid with a selenium atom. Human phagocytic cells were exposed to different aggregate species formed from the selenium-labelled Aβ fragment and its selenium-free analogue to examine the toxicity, uptake and distribution of the aggregates. The monomeric protein and the fully aggregated mature amyloid fibrils did not show significant levels of toxicity whereas aggregation species occurring earlier in the aggregation process were found to be highly cytotoxic, in agreement with previous studies on similar species. Cells exposed to the selenium-labelled aggregates were imaged using high angle annular dark field scanning transmission electron microscopy (HAADF-STEM), an electron microscopy technique in which only those electrons that are scattered to relatively high angles are used to generate an image. The majority of these electrons have undergone Rutherford scattering, the cross-section of which is dependent on Zn (n ~ 2). HAADF-STEM is therefore highly sensitive to local variations in atomic number. This technique has been used to visualise the selenium-labelled protein aggregates inside cells in two and three dimensions. The uptake and intracellular distributions of toxic and non-toxic aggregate species have been assessed and distinct differences have been observed correlating with the differences in toxicity

Advanced characterisation methods for the analysis of nanoformulations and extracellular vesicles

Nanomedicine represents a challenging and highly multidisciplinary research field, concerned with the development and study of nanoformulations for diagnostic and/or therapeutic purposes. The nano-sized particles of interest are increasingly complex. Their translational potential has been hampered by difficulties in their thorough characterisation. On the nano scale, small variations in size and composition can have large implications for their pharmacodynamic and pharmacokinetic behaviour. More precise techniques are therefore required to address these challenges. This thesis describes novel, advanced characterisation methods designed for the detailed study of single nanoparticles and their interaction and uptake behaviour with cells. A platform technology for Single Particle Automated Raman Trapping Analysis – SPARTA - was developed, capable of non-destructive, label-free and automated comprehensive single particle analysis. With the SPARTA system, the composition, functionalisation, size and dynamic reactions on the surface can be investigated in detail, of a wide variety of nanoparticles, through their Raman spectra. A further improved, custom designed SPARTA 2.0 platform was built, optimised for the analysis of complex biological particles, such as EVs. EVs represent a high potential as biomarkers, studied here in the context of breast cancer. The SPARTA 2.0 platform was able to resolve compositional differences between non-cancerous and cancer cell-derived EVs with excellent sensitivity and specificity. This highlights the possibility for development of new minimally invasive diagnostic approaches. In addition, a new imaging strategy for investigation of the EV-cellular interaction is presented, based on 3D Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM). FIB-SEM allows the generation of 3D models of the subcellular structure and visualisation of the cellular trafficking of nanoparticles. This represents a powerful new approach for investigating EV uptake. The methods developed in this thesis allow for the single particle-based analysis of a wide variety of nanoformulations and EVs, to aid in understanding their composition, applicability and cellular interactions.Open Acces