BackgroundMinor hallucinations (MH) affect 30-60% of patients with Parkinson's disease (PD), and are considered precursors to structured visual hallucinations and cognitive decline. While the link between structured visual hallucinations and dementia is well established, the neuropsychological correlates of MH in PD remain unclear; most studies finding no significant cognitive differences between patients with MH and those without any hallucinations.ObjectivesPresence hallucinations (PH) being among the most prevalent MH in PD, we used a robotic procedure delivering somatomotor conflicts inducing PH experimentally to investigate whether sensitivity to such robot-induced PH aids in detecting cognitive differences between patients with MH and without hallucinations.Methods31 PD patients with MH (PD-MH) and 37 without hallucinations (PD-nH) underwent neuropsychological assessment and the robotic procedure inducing PH. The sensitivity to report robot-induced PH was analyzed in relation to cognitive performance in neuropsychological tests.ResultsPD-MH patients reported more robot-induced PH than PD-nH patients, supporting previous findings. While both groups showed comparable performance in neuropsychological testing, we found a significant association between increased sensitivity to the PH-induction and poorer performance in frontal subcortical cognitive functions, in PD-MH patients, but not in PD-nH patients.ConclusionsThese findings demonstrate that sensitivity to robot-induced PH reveals a previously undetected link between MH and frontal subcortical cognitive deficits in PD, pointing to shared underlying mechanisms between executive dysfunction and somatomotor processes involved in MH. This approach offers a novel and clinically valuable means of identifying early cognitive vulnerability that assessments relying only on standard testing may overlook.

Blood biomarkers are rapidly becoming established for Alzheimer's Disease (AD) diagnosis. However, there is a need for more scalable tools to reach the 99% of individuals with early cognitive impairment who are not seen in specialist healthcare services. A recent study validated a capillary blood sampling technique to detect the p-tau217 and GFAP biomarkers. Here we used our PROTECT research study to show that these biomarkers, when collected using self-administered fingerprick tests, correlate well with venous blood biomarkers and with cognition and function in 174 people who were cognitively normal or who had mild cognitive impairment or AD. They can be used in combination with computerised cognitive testing to identify people with the highest risk of AD. The GFAP biomarker appears to be associated with vascular risk, unlike p-tau217. Patient feedback indicates high acceptability and usability of the capillary test method, giving confidence in the feasibility of this technology. The work suggests that capillary blood biomarkers could be used to enable triage of people with varying levels of risk of AD in clinical practice and for clinical trials, and could be used outside of clinical settings.

Super-resolution microscopy has emerged as a groundbreaking tool in microbiology, enabling the visualization of structures and molecules far below the diffraction limit of light. We provide an overview of the various techniques employed in super-resolution microscopy, including deterministic (scanning and structured illumination) and stochastic (single-molecule localization microscopy and fluctuation-based computation) methods, summarizing their respective advantages and limitations. Applications in microbiology are presented, including investigations of cellular processes, physiology, cell wall biology, and elucidation of bacterial toxin pathogenesis mechanisms at nanoscale resolution. New and emerging uses of super-resolution microscopy are also explored, focusing on addressing critical challenges in the field: pathogen-host interactions, antibiotic resistance, mode of action of bacterial toxins, and nanoscale detail of bacterial secretion systems and their translocated effectors. With the expanding accessibility of resources and increasing availability of open-source software to democratize data analysis, super-resolution microscopy is poised to revolutionize our understanding of the nanoscale world in microbiology, paving the way for new discoveries.

Most, if not all, cellular processes, exemplified by neuroexocytosis, rely on the precise interplay of specific molecules in both space and time. Super-resolution microscopy allows visualization of single-molecule trajectories far below the diffraction limit of visible light, which opens up possibilities for precise spatiotemporal analysis of these cellular processes. While a range of tools have been developed for assessing the spatial distribution of molecules in fixed cell data, the increasing sophistication of single particle tracking (spt) in live cells requires new approaches for spatiotemporal analysis, which can shed light on the nanoscale dynamics of molecular interaction and clustering. In this chapter, we will discuss a recently developed suite of novel tools-NAnoscale SpatioTemporal Indexing Clustering (NASTIC), which uses the overlap of molecular trajectory bounding boxes to establish interaction in space and time, and will describe a workflow to allow the user to use NASTIC to derive spatiotemporal metrics from their own super-resolved live cell trajectory data.

Single-molecule imaging is a technique of choice to investigate the dynamic nanoscale organization of proteins of interest, revealing a wealth of new information on how proteins perform their biological function. However, overexpression, which is often used in this context, can alter biological functions. It is therefore useful to implement tools to visualize and track endogenous proteins in living cells and observe their dynamic clustering and motion within their native environment. Herein, we describe two approaches: Fluorescent intrabody Localization Microscopy (FiLM) and gene-editing (CRISPR/Cas9) and provide a workflow including the design of plasmid backbones used to track endogenous proteins by either i) introducing nanobodies that target endogenous proteins to perform single-particle tracking or ii) gene-editing cells in culture to express tagged endogenous proteins at biologically relevant expression levels.

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are densely packed within the presynaptic bouton of neurons. The mechanisms by which SVs are clustered at the presynapse while dynamically organizing themselves into different SV pools with distinct fusion probabilities remain unknown. The study of SVs has historically been limited to ultrastructural studies of the presynapse. Examining the nanoscale dynamic organization of SVs in live neurons requires the use of innovative optical labelling approaches, super-resolution microscopy techniques, and appropriate stimulation paradigms that can mimic neuronal physiology. In this chapter, we discuss these aspects by highlighting the use of single-particle tracking photoactivated localization microscopy (sptPALM) to resolve the mobility and clustering of the total pool of SVs, Universal Point Accumulation Imaging in Nanoscale Topography (uPAINT) to study the mobility of SV proteins transiting on the plasma membrane, Dual-pulse subdiffractional Tracking of Internalized Molecules (DsdTIM) to simultaneously track the reserve and recycling pool of SVs and electrical field stimulation for depolarizing primary neurons.

Single-molecule localization microscopy (SMLM) has provided unique insight into the biophysical and nanoscale molecular mechanisms that underpin fundamental cellular functions. Nonetheless, due to technical limitations and challenges, much of the current work on real-time SMLM has relied heavily on in vitro or in cellulo model systems, which may not comprehensively reflect molecular dynamics in vivo. The current chapter acts as a guide for experimenters aiming to extend their research into in vivo or ex vivo SMLM, highlighting the obstacles and challenges that become apparent when attempting to elucidate the nanoscale cellular environment within complex multicellular organisms. Here, we describe our procedure for performing in vivo SMLM at the transgenic Drosophila melanogaster larval neuromuscular junction and provide insight into how to overcome these challenges. The protocol described allows direct visualization of molecules involved in synaptic communication within in vivo presynaptic nerve terminals and can be extended to other live multicellular organisms and systems.

In comparison to single-molecule localization microscopy (SMLM) of proteins located on the cell surface, SMLM of proteins within the nucleus requires fluorescent tags that are generally brighter, longer-lasting and (in the case of fluorescent ligands) membrane-permeable. Although fluorescent ligands typically have superior optical qualities compared to those of fluorescent protein (FP) tags, making them ideal candidates for nuclear SMLM, unlike FPs, they are unable to be expressed in cells as a fusion protein with the protein of interest. Self-labelling tags, on the other hand (e.g., HaloTag, SNAP-tag, and CLIP-tag), combine the ability to express a protein tag (e.g., HaloTag protein) as a fusion protein, with the enhanced optical qualities of externally added fluorescent dyes (e.g., dye-conjugated HaloTag ligand which covalently binds to the HaloTag protein). Excitation of only a subset of fluorescently labelled molecules is essential for achieving single-molecule resolution, and therefore SMLM of nuclear proteins requires a microscope setup capable of penetrating the nucleus with a thin beam that is powerful enough to produce an appropriate signal-to-noise ratio. Highly Inclined and Laminated Optical sheet (HILO) microscopy uses an oblique angle (just below that of Total Internal Reflection Fluorescence (TIRF)) to excite fluorescently labelled molecules within a thin, highly inclined plane, the signal of which can be amplified using a high-power TIRF filter.This chapter describes the following protocols: (i) seeding of cells into glass-bottom imaging dishes, (ii) transfection of cells with a HaloTag-tagged nuclear protein of interest, (iii) incubation of transfected cells with a fluorescent nuclear marker and a fluorescent HaloTag ligand, and (iv) single-molecule imaging of the fluorescently labelled HaloTag-tagged protein of interest in the nucleus of live cells using HILO microscopy with a high-powered TIRF filter.

Synaptic neurotransmission is an adaptive process in which synapses exhibit short- and long-term plasticity, dynamically strengthening or weakening their output in response to stimulation. Central to this fine-tuning is the reorganization of synaptic machinery and architecture, with nanoscale alterations in receptor clustering, vesicle trafficking and cytoskeletal rearrangements taking place over a short time frame. Traditionally, visualizing the dynamic organization of the synapse through confocal imaging has been challenging, considering the synapse's small size and the resolution limits imposed by light diffraction. The emergence of super-resolution imaging, and especially single-molecule localization microscopy (SMLM), over the last couple of decades, has allowed scientists to achieve nanoscale resolution and start unravelling the molecular underpinnings of synaptic transmission and plasticity in real-time. One such approach is single particle tracking Photo-Activated Localization Microscopy (sptPALM), which takes advantage of total internal reflection fluorescence (TIRF) and sparse, stochastic activation of fluorophores to achieve low spatiotemporal density labelling required to track single synaptic proteins in real time. In this chapter, we provide a conceptual breakdown of sptPALM: its key principles, implementation, and impact on the field of neurotransmission. Further, we detail a systematic protocol for performing sptPALM imaging of synaptic proteins in primary neuronal cultures. This technique allows the experimenter to determine the mobility, cluster patterns, and behavior of the synaptic proteins with nanoscale localization precision.

Fluorescence microscopy has become one of the most widely used tools in modern cell biology. By selectively labelling molecules of interest with fluorescent tags, researchers can visualize the structural organization, distribution, and clustering of proteins within their native cellular environment. Single-molecule localization microscopy (SMLM) has extended fluorescence imaging by enabling spatial resolution far beyond the diffraction limit of light. Among SMLM techniques, fixed Photoactivated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM), and Direct Stochastic Optical Reconstruction Microscopy (dSTORM) provide access to subcellular organization below the diffraction limit that is otherwise difficult to achieve through conventional or even intermediate super-resolution methods, such as Structured Illumination Microscopy (SIM) or Stimulated Emission Depletion microscopy (STED). These techniques attain super-resolution by temporally separating fluorophore emission events, localizing individual molecules with high precision, and assembling their positions into a reconstructed image. Despite the recent advances of newer super-resolution modalities, fixed PALM and (d)STORM remain widely used, due to their molecular specificity, accessibility, and broad applicability across diverse biological systems. This chapter briefly introduces the core principles of SMLM, situates PALM and (d)STORM within the evolution of super-resolution microscopy, and provides detailed protocols for the image acquisition and data analysis of fixed-cell PALM and dSTORM imaging in monolayer cultures, such as PC12 cells and primary hippocampal neurons. The workflow described here is compatible with standard SMLM platforms and uses Zeiss ZEN Black 2012 and Abbelight NEO software for reconstruction and analysis.

Super-resolution spinning-disk confocal microscopy with optical photon reassignment (SoRa) is an advanced imaging technique that extends fluorescence microscopy resolution beyond the diffraction limit without the need for specialized sample preparation or intensive computational processing. By enhancing both lateral and axial resolution while preserving the rapid imaging speed of conventional spinning-disk systems, SoRa is particularly well suited for live-cell imaging and high-throughput studies. In contrast to other super-resolution approaches, SoRa enables fast, 3D imaging with low phototoxicity, making it ideal for capturing dynamic cellular events in real time. This chapter highlights the use of SoRa for two key applications: (1) high-throughput screening, specifically for assessing drug effects on neuronal processes such as dendritic outgrowth, and (2) high-resolution 3D imaging of live-cells including imaging mitochondrial trafficking in neurons. SoRa's versatility, speed, and compatibility with standard dyes make it a powerful tool for a wide range of biological and drug discovery research.

Alzheimer's disease (AD) is a progressive and the most common neurodegenerative condition, having a deleterious effect on memory, eventually leading to death. In the recent past, gene therapy has emerged as a promising and revolutionary treatment for AD. This study demonstrated that nucleic acid-loaded nanoparticles which deliver small interfering RNA through lipid nanoparticles successfully reduced Alzheimer's disease-related symptoms in preclinical models by decreasing amyloid-β levels and enhancing cognitive abilities. However, every rose has its thorn, as the output of gene therapy is considerably hampered by the physiological barriers of the brain, which include the blood-brain barrier and the brain's extracellular matrix (ECM). For this reason, many researchers have modified the gene delivery technique by developing 'brain penetrating' NPs coated with components that can prevent sticking to the ECM. Moreover, to overcome the challenge of low transgene expression and reduced accumulation in the brain, even when delivered at high doses, scientists have proposed that injection/delivery of gene vectors directly into a specific area in the brain can achieve maximum therapeutic efficacy. Hence, this review focuses on the advancements and advantages of region-specific delivery of nucleic acid-loaded NPs for the effective therapeutic management of AD.

In epidemiological research, time-to-event outcomes, commonly referred to as survival outcomes, are a common subject of investigation. Here, we first describe the circumstances under which methods of survival analysis are necessary, review the roles of hazard functions, and discuss the limitations of hazard ratios for causal inference. Second, we explain how confounding and dependent censoring, which are common in observational studies, can be addressed by inverse probability weighting and parametric g-formula estimators. We emphasize that hazards serve as building blocks for estimating counterfactual risks. Third, we summarize recent developments in defining causal estimands in the presence of competing risks, including risk without eliminating competing events, net risk, and cumulative incidence under modified treatment. To illustrate how these challenges are addressed in practice, we revisit a recent clinical study on pharmacological interventions for the onset of dementia. We further empirically compare various estimands in the presence of competing events, including separable effects, through simulations. Our overall aim is to elucidate the need to move beyond routinely used methods of survival analysis, particularly the mere estimation of hazard ratios, if the goal is to draw causal inferences. This paper provides an overview of causal survival analysis, focusing on how confounding, dependent censoring, and competing risks can be addressed to estimate causal parameters of interest (counterfactual survival functions or counterfactual risks), which are interpretable and often meaningful for investigators.