Biology subjects

Manton, J. D.

Publications and source records attributed to Manton, J. D..

14 recordsLinked to original sources

bioRxiv2026

Index-agnostic oblique plane light sheet microscopy of centimetre-scale cleared tissues at subcellular resolution

We present cleared-tissue direct-view oblique plane microscopy (CtDvOPM), which enables optically sectioned subcellular resolution imaging of centimetre-scale tissues at high throughput over the full range of clearing media refractive indices (n = 1.33-1.56). CtDvOPM can image conventionally-mounted expanded, aqueous or non-aqueous cleared tissue samples at up to 2 {micro}m lateral by 14 {micro}m axial resolution over a 10.1 mm x 25.0 mm x 6.8 mm (x x y x z) sample volume without image tiling, at up to 400 million voxels per second.

biophysics↗

bioRxiv2025

Maximising imaging volumes of expanded tissues for inverted fluorescence microscopy

Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However, the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes, particularly due to the limited working distance of high-numerical aperture (NA) water immersion objectives. Here, we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations, we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems, which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity, we use an arrangement relying on an FEP film, Immersol W, water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution, supports large tile-scan acquisitions, and maintains image quality across depths exceeding 800 {micro}m. We validate the system by imaging 4x-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost, plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes.

biophysics↗

bioRxiv2025

Volumetric single-molecule tracking inside subcellular structures

The molecular interactions that underpin all cellular functions depend on molecular motion within three-dimensional environments. Large depth-of-field single-molecule localization microscopy (3D-SMLM) methods facilitate these measurements, but their increased optical complexity and bespoke post-processing pipelines can sacrifice important cellular context. Here we combine single-molecule light-field microscopy (SMLFM) with widefield Fourier light-field microscopy for correlative volumetric organelle imaging. The instantaneous acquisition of subcellular volumes improves the sensitivity of molecular organization and diffusion measurements through the rejection of non-specific localizations. We first demonstrate our approach by measuring the molecular organization of a nuclear-localized HaloTag protein relative to cell nuclei. Next, we characterize the molecular diffusion of the soluble protein, calreticulin, in the context of 1-antitrypsin deficiency, which revealed an increase in heterogeneous motion within endoplasmic reticulum inclusions.

biophysics↗

bioRxiv2025

A deconvolution solution to aliasing confusion in apparently undersampled oblique plane microscopy

In oblique plane microscopy, a remote refocussing system is used to create an aberration-free 3D image of the object, from which an inclined plane is sampled using a tilted imaging system. As a result, unlike in conventional microscopes, the point spread function (PSF) and optical transfer function (OTF) are tilted with respect to the optical axis of the imaging system. This suggests that a small sample-scanning step size is required to satisfy the Nyquist-Shannon sampling criterion. Recently, we have shown that a careful choice of apparent undersampling leads to aliased OTF copies that can be separated and stitched together in Fourier space to form a properly sampled OTF. In our demonstrations, this led to acquisition speed gains of between twoand four-fold. Here, we introduce a deconvolution-based method to reconstruct properly sampled volumes from apparently undersampled datasets that outperforms our previous Fourier-stitching approach.

biophysics↗

bioRxiv2025

Direct-view oblique plane microscopy

The ability to rapidly image mesoscopic samples is critically important for many areas of biological research. Owing to its high through-put and gentle, volumetric imaging nature, light-sheet fluorescence microscopy (LSFM) is an attractive modality to image such samples. However, the orthogonal dual-objective geometry of LSFM makes sample mounting challenging. Oblique plane microscopy (OPM) circumvents these issues by achieving light-sheet imaging through a single objective near the sample, with a further two objectives acting to image a tilted, oblique plane in the specimen. However, at low magnification, as required for mesoscopic imaging, conventional OPM systems suffer from a complicated and inefficient light path and/or a limited numerical aperture. Here we present a simple method for mesoscopic OPM that enables efficient light collection at any numerical aperture. By placing the camera directly in the remote space, the oblique plane can be imaged without the need for a third microscope system. Using a commercially available camera with 1.4 {micro}m pixels, we demonstrate imaging over a 5.3 x 3 x 2.6 mm3 field of view with a lateral resolution of [~]2 {micro}m and axial resolution of [~]22 {micro}m. Our technique is then used to image a 4x-expanded brain organoid.

biophysics↗

bioRxiv2024

Increasing the acquisition speed in oblique plane microscopy via Aliasing

Oblique plane microcopy (OPM), a variant of light-sheet fluorescence microscopy (LSFM), enables rapid volumetric imaging without mechanically scanning the sample or an objective. In an OPM, the sample space is mapped to a distortion free image space via remote focusing, and the oblique light-sheet plane is mapped via a tilted tertiary imaging system onto a camera. As a result, the 3D point-spread function and optical transfer function are tilted to the optical axis of the tertiary imaging system. To satisfy Nyquist sampling, small scanning steps are required to encompass the tilted 3D OTF, slowing down acquisition and increasing sample exposure. Here we show that a judicious amount of under-sampling can lead to a form of aliasing in OPM that can be recovered without a loss of spatial resolution or introducing artifacts. The resulting speed gain depends on the optical parameters of the system and can reach 2-4-fold in our demonstrations. We leverage this speed gain for rapid subcellular 3D imaging of mitochondrial dynamics.

biophysics↗

bioRxiv2024

Multispectral live-cell imaging with uncompromised spatiotemporal resolution

Multispectral imaging is an established method to extend the number of colours usable in fluorescence imaging beyond the typical limit of three or four, but standard approaches are poorly suited to live-cell imaging. We introduce an approach for multispectral imaging in live cells, comprising an iterative spectral unmixing algorithm and eight channel camera-based image acquisition hardware. This enables the accurate unmixing of low signal-to-noise ratio datasets, typical of live-cell imaging, captured at video rates. We use this approach on a commercial spinning disk confocal microscope and a home-built oblique plane light sheet microscope to image 1-7 spectrally distinct fluorophore species simultaneously, using both fluorescent protein fusions and small-molecule dyes. We further use de novo designed protein-binding proteins (minibinders), labelled with organic fluorophores, and use these in combination with our multispectral imaging approach to study the endosomal trafficking of cell-surface receptors at endogenous levels.

biophysics↗

bioRxiv2024

Synthesis of arbitrary interference patterns using a single galvanometric mirror, and its application to Structured Illumination Microscopy

We present a new interferometer concept called SWIFT, able to project arbitrary interference patterns constructed from small numbers of plane waves. SWIFT can control each plane waves orientation, intensity, polarization and phase using just a single galvanometric mirror. We demonstrate the application of SWIFT to both 2D and 3D Structured Illumination Microscopy, characterizing performance on fluorescent nanoparticles and iFluor 488 phalloidin-stained U-2 OS cells.

bioengineering↗

bioRxiv2022

Induction of cortical Par complex polarity by designed proteins causes cytoskeletal symmetry breaking in unpolarized mammalian cells

Polarized cells rely on a polarized cytoskeleton for polarized trafficking, oriented migration and spindle orientation during asymmetric cell division. While cytoskeleton remodeling machineries have been extensively characterized at the molecular level, how polarity signaling at the cortex controls remodeling of the cytoskeleton in the cytosol remains elusive. In particular, how the Par complex, the conserved mastermind of polarity during asymmetric cell division, gets assembled and functions is not understood at the molecular level. Here, we dissected the logic of the Par complex pathway by capitalizing on designed proteins able to induce spontaneous symmetry breaking of the cortex in populations of naive, unpolarized cells. We found that the primary kinetic barrier to Par complex assembly is the relief of Par6 autoinhibition, and that inducing Par complex cortical polarity was sufficient to induce two key hallmarks of asymmetric cell division in unpolarized cells: spindle orientation and central spindle asymmetry. These two outputs of the Par complex are separately controlled: spindle orientation is determined by Par3 and does not require the kinase activity of aPKC, while central spindle asymmetry solely depends on an asymmetric activity of aPKC at the cortex. Our work shows how polarity information flows between the cortex and the cytosol despite its diffusive nature, and paves the way towards induction of asymmetric cell division in cultured cells.

cell biology↗

bioRxiv2020

Real-Time Multi-Angle Projection Imaging of Biological Dynamics

We introduce a cost-effective and easily implemented scan unit which enables any camera-based microscope to perform projection imaging from diverse viewing angles. We demonstrate this capability on Lattice Light-Sheet and Oblique Plane Microscopy by rapidly delivering projection images with an uncompromised lateral resolution and high optical contrast. By imaging the sample from one or multiple perspectives, our method enables visualization of rapid biological processes, real time stereoscopic imaging as well as three-dimensional particle localization throughout a cellular volume from just two images. Furthermore, because our projection imaging technique provides intuitive three-dimensional renderings in real-time, it improves microscope usability, allows users to more-readily optimize instrument performance and identify biological phenomena of interest on-the-fly, while also reducing data overhead by a factor of >100. We leverage our rapid projection method to image cancer cell morpho-dynamics and calcium signaling in cultured neurons, to perform three-dimensional localization of genetically encoded nanoparticles, as well as to image orthogonal views of an embryonic Zebrafish heart simultaneously.

biophysics↗

bioRxiv2019

Spatiotemporal analysis of host cell modification during herpes simplex virus 1 replication

Herpesviruses are large and complex viruses, and have a long evolutionary history with their host species. One poorly understood, but important, area in virus-host interaction is the extensive remodelling of intracellular organelles and morphology that occurs during active viral replication. We have constructed a recombinant reporter virus that allowed us to classify four distinct stages in the infection cycle of herpes simplex virus 1. Single-cell analysis of the impact of the viral load on replication dynamics demonstrates the utility of this times-tamping method for tracking the infection cycle. High-resolution microscopy analysis of cellular structures in live and fixed cells in concert with our dual-fluorescent reporter virus have enabled us to generate a detailed overview over the spatial and temporal organisation of the cytoskeleton and organelles during viral replication. This provides the first systems-level analysis of the morphological changes involved in viral replication.

microbiology↗

bioRxiv2019

Concepts for structured illumination microscopy with extended axial resolution through mirrored illumination

Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.

bioengineering↗

bioRxiv2019

Intra-mitochondrial protein degradationalleviates alpha-synuclein seeding and Abeta 42 aggregation

Mitochondria have long been implicated in Parkinsons disease (PD), however, it is not clear how mitochondrial impairment and alpha-synuclein pathology are coupled. We report here that intra-mitochondrial protein homeostasis plays a major role in alpha-synuclein fibril elongation, as interference with intra-mitochondrial proteases and mitochondrial protein import significantly aggravate alpha-synuclein aggregation. In contrast, direct inhibition of mitochondrial complex I, increase in intracellular calcium concentration or formation of reactive oxygen species (ROS), all of which have been associated with mitochondrial stress, did not affect alpha-synuclein pathology. We further demonstrate that similar mechanisms are involved in Amyloid {beta} 1-42 (A{beta}42) aggregation, suggesting that mitochondria are directly capable of influencing cytosolic protein homeostasis of aggregation-prone proteins.

neuroscience↗

bioRxiv2018

Functional and Anatomical Specificity in a Higher Olfactory Centre

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. For example, in insects, second order olfactory projection neurons target both the mushroom body, which is required for learning, and the lateral horn (LH), which has been proposed to mediate innate olfactory behavior. Mushroom body neurons encode odors in a sparse population code, which does not appear stereotyped across animals. In contrast the functional principles of odor coding in the LH remain poorly understood. We have carried out a comprehensive anatomical analysis of the Drosophila LH, counting ~1400 neurons; combining genetic driver lines, anatomical and functional criteria, we identify 165 LHN cell types. We then show that genetically labeled LHNs have stereotyped odor responses across animals for 33 of these cell types. LHN tuning can be ultra-sparse (1/40 odors tested), but on average single LHNs respond to three times more odors than single projection neurons. This difference can be rationalized by our observation that LHNs are better odor categorizers, likely due to pooling of input projection neurons responding to different odors of the same category. Our results reveal some of the principles by which a higher sensory processing area can extract innate behavioral significance from sensory stimuli.

neuroscience↗