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Compact and contactless reflectance confocal microscope for neurosurgery
Visual guidance at the cellular level during neurosurgical procedures is essential for complete tumour resection. We present a compact reflectance confocal microscope with a 20 mm working distance that provided <1.2 μm spatial resolution over a 600 μm × 600 μm field of view in the near-infrared region. A physical footprint of 200 mm × 550 mm was achieved using only standard off-the-shelf components. Theoretical performance of the optical design was first evaluated via commercial Zemax software. Then three specimens from rodents: fixed brain, frozen calvaria and live hippocampal slices, were used to experimentally assess system capability and robustness. Results show great potential for the proposed system to be translated into use as a next generation label-free and contactless neurosurgical microscope.
z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics.
Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.
Electrically-tunable positioning of topological defects in liquid crystals.
Topological defects are a consequence of broken symmetry in ordered systems and are important for understanding a wide variety of phenomena in physics. In liquid crystals (LCs), defects exist as points of discontinuous order in the vector field that describes the average orientation of the molecules in space and are crucial for explaining the fundamental behaviour and properties of these mesophases. Recently, LC defects have also been explored from the perspective of technological applications including self-assembly of nanomaterials, optical-vortex generation and in tunable plasmonic metamaterials. Here, we demonstrate the fabrication and stabilisation of electrically-tunable defects in an LC device using two-photon polymerisation and explore the dynamic behaviour of defects when confined by polymer structures laser-written in topologically discontinuous states. We anticipate that our defect fabrication technique will enable the realisation of tunable, 3D, reconfigurable LC templates towards nanoparticle self-assembly, tunable metamaterials and next-generation spatial light modulators for light-shaping.
Somatostatin secretion by Na+-dependent Ca2+-induced Ca2+ release in pancreatic delta-cells.
Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing β- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.
Reduced somatostatin signalling leads to hypersecretion of glucagon in mice fed a high-fat diet.
OBJECTIVES: Elevated plasma glucagon is an early symptom of diabetes, occurring in subjects with impaired glucose regulation. Here, we explored alpha-cell function in female mice fed a high-fat diet (HFD). METHODS: Female mice expressing the Ca2+ indicator GCaMP3 specifically in alpha-cells were fed a high-fat or control (CTL) diet. We then conducted in vivo phenotyping of these mice, as well as experiments on isolated (ex vivo) islets and in the in situ perfused pancreas. RESULTS: In HFD-fed mice, fed plasma glucagon levels were increased and glucagon secretion from isolated islets and in the perfused mouse pancreas was also elevated. In mice fed a CTL diet, increasing glucose reduced intracellular Ca2+ ([Ca2+]i) oscillation frequency and amplitude. This effect was also observed in HFD mice; however, both the frequency and amplitude of the [Ca2+]i oscillations were higher than those in CTL alpha-cells. Given that alpha-cells are under strong paracrine control from neighbouring somatostatin-secreting delta-cells, we hypothesised that this elevation of alpha-cell output was due to a lack of somatostatin (SST) secretion. Indeed, SST secretion in isolated islets from HFD-fed mice was reduced but exogenous SST also failed to suppress glucagon secretion and [Ca2+]i activity from HFD alpha-cells, in contrast to observations in CTL mice. CONCLUSIONS: These findings suggest that reduced delta-cell function, combined with intrinsic changes in alpha-cells including sensitivity to somatostatin, accounts for the hyperglucagonaemia in mice fed a HFD.
Validation of the foot profile score.
BACKGROUND: There are numerous static measures of foot posture but there is no published score of dynamic foot motion. Three-dimensional gait analysis can include a multi-segment foot model like the Oxford Foot Model (OFM) to comprehensively quantify foot kinematic deviations across the gait cycle but it lacks an overall score, like the Gait Profile score (GPS), used to summarize the quality of lower extremity motion. RESEARCH QUESTION: This paper introduces the Foot Profile Score (FPS), a single number, analogous to the GPS but based on kinematic data of the OFM. The aim of this study is to validate the FPS by studying its properties and design, and analyse it against a clinical assessment of foot deformity. METHODS: Concurrent validity was established for the FPS analysing the relationship with Clinical Foot Deformity Score (CFDS) in 60 subjects with a condition affecting the lower limbs globally Content validity was established for the six Foot Variable Scores (FVS) that make up the FPS using a multiple regression of the CFDS on the 6 FVS in the 60 subjects. Predictive validity was established analysing the relationship of the FPS and GPS comparing 60 global involvement subjects with 60 subjects with isolated foot deformity. RESULTS: Pearson correlation between the FPS and CFDS was significant at 0.62 (p < 0.001). Each element of FVS contributes positively to predicting the CFDS with R2 = 0.456 (p < 0.001). FPS contributed independently to the prediction of CFDS (t = 3.9, p < 0.001). The correlation between the GPS and FPS in the global involvement group was significant at r = 0.64 (p < 0.001), while there was no correlation found with r = 0.08 (p = 0.54) in the foot deformity group. SIGNIFICANCE: The FPS is the first validated score of dynamic foot motion.
Multi-scale sensorless adaptive optics: application to stimulated emission depletion microscopy.
Sensorless adaptive optics is commonly used to compensate specimen-induced aberrations in high-resolution fluorescence microscopy, but requires a bespoke approach to detect aberrations in different microscopy techniques, which hinders its widespread adoption. To overcome this limitation, we propose using wavelet analysis to quantify the loss of resolution due to the aberrations in microscope images. By examining the variations of the wavelet coefficients at different scales, we are able to establish a multi-valued image quality metric that can be successfully deployed in different microscopy techniques. To corroborate our arguments, we provide experimental verification of our method by performing aberration correction experiments in both confocal and STED microscopy using three different specimens.
Anisoplanatic adaptive optics in parallelized laser scanning microscopy.
Inhomogeneities in the refractive index of a biological microscopy sample can introduce phase aberrations, severely impairing the quality of images. Adaptive optics can be employed to correct for phase aberrations and improve image quality. However, conventional adaptive optics can only correct a single phase aberration for the whole field of view (isoplanatic correction) while, due to the highly heterogeneous nature of biological tissues, the sample induced aberrations in microscopy often vary throughout the field of view (anisoplanatic aberration), limiting significantly the effectiveness of adaptive optics. This paper reports on a new approach for aberration correction in laser scanning confocal microscopy, in which a spatial light modulator is used to generate multiple excitation points in the sample to simultaneously scan different portions of the field of view with completely independent correction, achieving anisoplanatic compensation of sample induced aberrations, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions. The method was tested in whole Drosophila brains and in larval Zebrafish, each showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.
Behavioral flexibility is associated with changes in structure and function distributed across a frontal cortical network in macaques.
One of the most influential accounts of central orbitofrontal cortex-that it mediates behavioral flexibility-has been challenged by the finding that discrimination reversal in macaques, the classic test of behavioral flexibility, is unaffected when lesions are made by excitotoxin injection rather than aspiration. This suggests that the critical brain circuit mediating behavioral flexibility in reversal tasks lies beyond the central orbitofrontal cortex. To determine its identity, a group of nine macaques were taught discrimination reversal learning tasks, and its impact on gray matter was measured. Magnetic resonance imaging scans were taken before and after learning and compared with scans from two control groups, each comprising 10 animals. One control group learned discrimination tasks that were similar but lacked any reversal component, and the other control group engaged in no learning. Gray matter changes were prominent in posterior orbitofrontal cortex/anterior insula but were also found in three other frontal cortical regions: lateral orbitofrontal cortex (orbital part of area 12 [12o]), cingulate cortex, and lateral prefrontal cortex. In a second analysis, neural activity in posterior orbitofrontal cortex/anterior insula was measured at rest, and its pattern of coupling with the other frontal cortical regions was assessed. Activity coupling increased significantly in the reversal learning group in comparison with controls. In a final set of experiments, we used similar structural imaging procedures and analyses to demonstrate that aspiration lesion of central orbitofrontal cortex, of the type known to affect discrimination learning, affected structure and activity in the same frontal cortical circuit. The results identify a distributed frontal cortical circuit associated with behavioral flexibility.
Cell-Penetrating Peptide Conjugates of Steric Blocking Oligonucleotides as Therapeutics for Neuromuscular Diseases from a Historical Perspective to Current Prospects of Treatment.
The review starts with a historical perspective of the achievements of the Gait group in synthesis of oligonucleotides (ONs) and their peptide conjugates toward the award of the 2017 Oligonucleotide Therapeutic Society Lifetime Achievement Award. This acts as a prelude to the rewarding collaborative studies in the Gait and Wood research groups aimed toward the enhanced delivery of charge neutral ON drugs and the development of a series of Arg-rich cell-penetrating peptides called Pip (peptide nucleic acid/phosphorodiamidate morpholino oligonucleotide [PNA/PMO] internalization peptides) as conjugates of such ONs. In this review we concentrate on these developments toward the treatment of the neuromuscular diseases Duchenne muscular dystrophy and spinal muscular atrophy toward a platform technology for the enhancement of cellular and in vivo delivery suitable for widespread use as neuromuscular and neurodegenerative ON drugs.
CPP-directed oligonucleotide exon skipping in animal models of Duchenne muscular dystrophy.
Antisense oligonucleotides (AOs) are effective splice switching agents and have potential as therapeutics via the exclusion or inclusion of specific target gene exons to ameliorate and modify disease progression. The leading example is Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disease, where AO-mediated skipping of specific DMD gene exons can restore the disrupted DMD open reading frame, leading to the production of functional dystrophin protein and ameliorate the DMD phenotype in animal models. Clinical proof-of-concept has recently been shown in two successful, independent Phase I clinical trials. These trials both followed local intramuscular treatments, and the challenge now is to develop and test systemic protocols, which will be required for treatment-aimed disease modification. Recently, a number of groups have demonstrated the promise of AOs directly conjugated to cell-penetrating peptides (CPPs) as having significant potential for systemic delivery and therapeutic correction in DMD animal models. Here, we review the background to this work and describe in detail the experimental protocols used in studies aimed at investigating CPP-conjugated AOs as systemic splice correcting agents in animal models of DMD.
Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology.
BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. METHODS: Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. RESULTS: The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. CONCLUSIONS: Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.
Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice.
Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics.