Biology subjects

Needham, L.-M.

Publications and source records attributed to Needham, L.-M..

8 recordsLinked to original sources

bioRxiv2024

Vortex light field microscopy: 3D spectral single-molecule imaging with a twist

We introduce vortex light field microscopy (VLFM), a novel method for snapshot 3D spectral single-molecule localization microscopy. Inspired by the azimuthal phase profile of optical vortices, we place an azimuthally oriented prism array immediately after the microlens array in a Fourier light field microscope (FLFM). This innovative arrangement causes the axial position and spectral peak for a point emitter to be encoded in the radial and azimuthal displacement of point-spread-function (PSF) respectively. This enables simultaneous detection of 3D position and emission peak of individual fluorophores with 25 nm spatial precision and 3 nm spectral precision over a 4 m depth of field (DOF). We illustrate the spectral scalability of our method by performing four-color 3D single particle tracking of freely diffusing fluorescent beads, and two-color 3D dSTORM imaging of microtubules and mitochondria in fixed COS-7 cells, without the need for spectrally distinct fluorophores.

biophysics↗

bioRxiv2024

Large-scale visualisation of α-synuclein oligomers in Parkinson's disease brain tissue

Parkinsons disease (PD) is a common neurodegenerative condition characterised by the presence in the brain of large intraneuronal aggregates, known as Lewy bodies and Lewy neurites, containing fibrillar -synuclein. According to the amyloid hypothesis, these large end-stage species form from smaller soluble protein assemblies, often termed oligomers, which are proposed as early drivers of pathogenesis. To date, however, this hypothesis has remained controversial, at least in part because it has not been possible to directly visualise oligomeric aggregates in human brain tissue. Therefore, their presence, abundance and distributions have remained elusive. Here, we present ASA-PD (Advanced Sensing of Aggregates - Parkinsons Disease), an imaging method to generate large-scale -synuclein oligomer maps in post-mortem human brain tissue. We combined autofluorescence suppression with single-molecule fluorescence methods, which together, enable the detection of nanoscale -synuclein aggregates. To demonstrate the utility of this platform, we captured [~]1.2 million oligomers from the anterior cingulate cortex in human post-mortem brain samples from PD and healthy control patients. Our data revealed a specific subpopulation of nanoscale oligomers that represent an early hallmark of the proteinopathy that underlies PD. We anticipate that quantitative information about oligomer distributions provided by ASA-PD will enable mechanistic studies to reveal the pathological processes caused by -synuclein aggregation.

neuroscience↗

bioRxiv2023

Label-free observation of individual solution phase molecules

The vast majority of chemistry and biology occurs in solution, and new label-free analytical techniques that can help resolve solution-phase complexity at the single-molecule level can provide new microscopic perspectives of unprecedented detail. Here, we use the increased light-molecule interactions in high-finesse fiber Fabry-Perot microcavities to detect individual biomolecules as small as 1.2 kDa with signal-to-noise ratios >100, even as the molecules are freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of sub-populations in mixed samples. Strikingly, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight can also be resolved. Detection is based on a novel molecular velocity filtering and dynamic thermal priming mechanism leveraging both photo-thermal bistability and Pound-Drever-Hall cavity locking. This technology holds broad potential for applications in life and chemical sciences and represents a major advancement in label-free in vitro single-molecule techniques.

biophysics↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2020

Wild-type sTREM2 blocks Aβ aggregation and neurotoxicity, while the Alzheimer's R47H mutant does the opposite

Missense mutations (e.g. R47H) of the microglial receptor TREM2 increase risk of Alzheimers disease (AD), and the soluble ectodomain of wild-type TREM2 (sTREM2) appears to protect in vivo, but the underlying mechanisms are unclear. We show that A{beta} oligomers bind to TREM2, inducing shedding of sTREM2. Wild-type sTREM2 inhibits A{beta} oligomerization, fibrillization and neurotoxicity, and disaggregates preformed A{beta} oligomers and protofibrils. In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric A{beta}, but rather promotes A{beta} protofibril formation and neurotoxicity. Thus, in addition to mediating phagocytosis, wild-type TREM2 may protect against amyloid pathology by A{beta}-induced release of sTREM2 that blocks A{beta} aggregation and neurotoxicity; while R47H sTREM2 promotes A{beta} aggregation into neurotoxic forms, which may explain why the R47H variant gene increases AD risk several fold.

biochemistry↗

bioRxiv2020

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local regions of the genome to which it binds.

cell biology↗

bioRxiv2020

vLUME: 3D Virtual Reality for Single-molecule Localization Microscopy

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established1,2 and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching3. While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced volume) a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive, Supplementary Video 1). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.

biophysics↗

bioRxiv2019

ThX - A next-generation probe for the early detection of amyloid aggregates

Neurodegenerative diseases such as Alzheimers and Parkinsons are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, we demonstrate that ThX can be used to detect a distinct oligomeric species, not observed via traditional ThT imaging.

biophysics↗