Biology subjects

Lee, S. F.

Publications and source records attributed to Lee, S. F..

At least 19 recordsLinked to original sources

bioRxiv2025

Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells

Maintenance of naive pluripotency requires core transcription factors (TFs) like SOX2 and auxiliary TFs like NANOG, yet molecular mechanisms governing their intra-nuclear dynamics and DNA binding interactions remain unclear. Here, using high-density 3D single-molecule light-field microscopy combined with novel spatiotemporal analysis pipelines, we track SOX2 and NANOG dynamics in live cells. Despite lower protein abundance, NANOG displays a similar chromatin-bound fraction to SOX2. This arises partially because, while both TFs undergo frequent transient non-specific binding interactions ([~]0.5-0.7s), NANOG exhibits more stable specific binding ([~]25s vs [~]16s). Both TFs also assemble into phase-separated domains of [~]400 nm containing both freely diffusing and chromatin-bound proteins, which further influences their dynamics. Strikingly, NANOGs protein-protein interaction domain markedly increases chromatin residence time (>5-fold) and the size of these phase-separated domains. Our work uncovers how NANOG and SOX2 stabilise gene regulatory networks that maintain naive pluripotency while providing quantitative pipelines for dissecting spatiotemporal TF dynamics.

cell biology↗

bioRxiv2025

SMOLM-LFM: ratiometric single molecule orientation without polarizers

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the methods potential and practical limitations.

bioengineering↗

bioRxiv2025

Nanoscale spatial-omics via contrastive embedding of single-molecule localisation data

Omics approaches have revolutionised biology, and cells can now be routinely characterised on the genomic, transcriptomic and proteomic levels. However, there is an additional pillar; the (nanoscale) spatial organisation of molecules in the cell - information now accessible through super-resolution microscopy. We present a contrastive learning framework for nanoscale spatial-omics that embeds single-molecule localisation microscopy data into a latent space representing protein architecture directly to enabling comparative analysis. Using simulated and experimental data, we demonstrate its ability to enable new bioanalysis capabilities including assessing changes to cellular nanoscale architecture arising from pharmacological treatments, cell type, fluorophore selection or data-processing workflows. The approach supports downstream tasks such as clustering proteins by nanoscale organisation, mapping dose-response trajectories and identifying batch effects in replicate datasets, establishing contrastive learning as a scalable foundation for nanoscale spatial-omics and providing a platform for comparative phenotyping, quality control, and hypothesis generation.

bioinformatics↗

bioRxiv2025

Neurodegeneration emerges at a cellular tipping point between protein accumulation and removal.

Protein aggregates are a pathological hallmark across neurodegenerative diseases. Yet, the disconnect between molecular-level aggregation and the emergence of disease severely limits mechanistic understanding of neurodegeneration. Here, we bridge this disconnect by showing that a cellular tipping point emerges as a universal feature across diseases from the competition between aggregate accumulation and removal. We map the resulting cellular phase transition with our high-throughput live-cell assay, measuring the tipping point that separates healthy cells from those with large aggregate loads. Using super-resolution imaging of brain tissue from Alzheimers and Parkinsons disease, we quantify how the balance of accumulation and removal is shifted in disease. We validate our framework by predicting how designed aggregation inhibitors shift the tipping point to restore cellular homeostasis. Our results provide a mechanistic framework connecting molecular-level aggregation to disease, paving the way for a quantitative, unified understanding of neurodegeneration and enabling predictions of therapeutic efficacy.

neuroscience↗

bioRxiv2025

T-cell signaling relies on partial CD45-exclusion at sub-micron sized cellular contacts

How cell contact initiates T-cell activation is uncertain. The local exclusion of the receptor-type protein tyrosine phosphatase CD45 at cell contacts is believed to trigger immune receptor signaling but this is yet to be observed for T cells interacting with authentic cellular targets. Here, quantitative imaging of T cells interacting with tumor cells presenting either native or clinically relevant bi-specific TCR ligands, revealed that they form multiple sub-micron sized close contacts with their targets. The contacts were stabilised by the adhesion protein CD2, but efficient ligand detection required both CD2 and integrin ligation. CD45 was excluded from close contacts at the time of ZAP70 recruitment and signaling, but only partially (30- 40%). A single-cell, mass cytometric analysis showed that this change in kinase/phosphatase activity provoked strong T-cell activation and potent cytotoxicity via very small changes in signaling fluxes. Spatial stochastic simulations suggested that the proximal T-cell signaling network is optimised for efficient antigen discrimination in the setting of partial CD45 exclusion. Our work re-frames early T-cell activation as a process initiated by relatively subtle changes in kinase/phosphatase activity acting on small numbers of signaling effectors at minute cellular contacts.

immunology↗

bioRxiv2025

Microglial activation and alpha-synuclein oligomers drive the early inflammatory phase of Parkinson's disease

Parkinsons disease (PD) is characterised by insoluble -synuclein (Syn) aggregates in Lewy bodies (LBs) within the substantia nigra, with cortical pathology appearing as the disease progresses. Late-stage LB deposition, cellular stress, and neuronal loss obscure disease-driving events, we therefore performed multi-regional transcriptomic and aggregate profiling in early-midstage PD brains (Braak 3-4), where cortical regions are pathologically unaffected. We report neuroimmune activation as an early PD feature, characterised by the expansion of a high-SNCA-expressing microglial state. This robust immune signature occurs prior to LB formation, but is associated with oligomeric Syn within cortical microglia. In hiPSC-derived microglia, both endogenous Syn oligomerisation, and exogenous oligomer uptake, trigger transcriptional reprogramming, characterised by interferon-driven inflammation, antigen presentation, and mitochondrial suppression, closely mirroring the early PD brain. These findings describe mechanisms by which Syn oligomerisation potently initiates early neuroinflammation, highlighting a critical interplay between proteinopathy and immune activation at the earliest stages of disease.

neuroscience↗

bioRxiv2025

α-Synuclein driven cell susceptibility in Parkinson's disease

Early cellular events in Parkinsons disease (PD) remain elusive. While aggregation of -synuclein (Syn) into Lewy bodies marks advanced pathology, smaller Syn oligomers have been implicated in prodromal stages. Here we map Syn oligomers at single-particle resolution in post-mortem brain tissue from Braak stage 3/4 PD cases and matched controls. Quantitative imaging of 9,882 neurons across four regions captured over 112 million Syn oligomers. Mean intracellular -Syn burden was unchanged between groups, but PD samples contained a higher fraction of neurons whose oligomer load exceeded a specific aggregation threshold. We term these aggregation-susceptible cells (ASCs). ASC enrichment in vulnerable regions supports a population-level model in which early pathology arises from a stochastic shift in cellular composition rather than altered Syn aggregation kinetics. This human-tissue, large-scale dataset provides a quantitative framework for detecting ASCs and for testing population-level interventions in PD and related proteinopathies.

neuroscience↗

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↗

bioRxiv2024

3D super-resolution imaging of PSD95 reveals an abundance of diffuse protein supercomplexes in the mouse brain

PSD95 is an abundant scaffolding protein that assembles multiprotein complexes controlling synaptic physiology and behavior. Confocal microscopy has previously shown that PSD95 is enriched in the postsynaptic terminals of excitatory synapses and 2D super-resolution microscopy further revealed that it forms nanoclusters. In this study, we utilized 3D super-resolution microscopy to examine the nanoarchitecture of PSD95 in the mouse brain, characterizing over 8 million molecules. While we were able to identify structural subtypes previously reported, imaging in 3D allowed us to classify these with higher accuracy. Furthermore, 3D super-resolution microscopy enabled the quantification of protein levels, revealing an abundance of PSD95 molecules existed outside of synapses as a diffuse population of supercomplexes, containing multiple copies of PSD95. Further analysis of the supercomplexes containing two units identified two populations: one that had PSD95 molecules separated by 39 {+/-} 2 nm, and a second with a separation of 94 {+/-} 27 nm. These results suggest that PSD95 supercomplexes containing multiple protein copies assemble outside the synapse and then integrate into the synapse to form a supramolecular nanocluster architecture.

biophysics↗

bioRxiv2024

Nano-org, a functional resource for single-molecule localisation microscopy data

We present a publicly accessible, curated, and functional resource, termed "nano-org", containing single-molecule localisation microscopy (SMLM) data representing the nanoscale distributions of proteins in cells. Nano-org is searchable by comparing the statistical similarity of the datasets it contains. This unique functionality allows the resource to be used to understand the relationships of nanoscale architectures between proteins, cell types or conditions, enabling a new field of spatial nano-omics.

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

RASP: Optimal single fluorescent puncta detection in complex cellular backgrounds

Super-resolution and single-molecule microscopy are increasingly applied to complex biological systems. A major challenge of this approach is that fluorescent puncta must be detected in the low signal, high noise, heterogeneous background environments of cells and tissue. We present RASP, Radiality Analysis of Single Puncta, a bioimaging-segmentation method that solves this problem. RASP removes false positive puncta that other analysis methods detect, and detects features over a broad range of spatial scales: from single proteins to complex cell phenotypes. RASP outperforms the state-of-the-art in precision and speed, using image gradients to separate Gaussian-shaped objects from background. We demonstrate RASPs power by showing it can extract spatial correlations between microglia, neurons, and -synuclein oligomers in the human brain. This sensitive, computationally efficient approach enables fluorescent puncta and cellular features to be distinguished in cellular and tissue environments with a sensitivity down to the level of the single protein.

biophysics↗

bioRxiv2023

Single-molecule digital sizing of proteins in solution

Proteins constitute the molecular machinery of life and exert their biological function by interacting with other proteins, as well as by assembling into biomolecular complexes and higher order structures. Characterizing the sizes, interactions, and assembly states of proteins is thus key for understanding the normal functional behavior of proteins and for elucidating aberrant processes and interactions that can lead to dysfunction and disease. However, the physical characterization of proteins has remained a challenging problem due to the inherent compositional heterogeneity of protein mixtures as well as the polydisperse nature of protein complexes. Here, we address this challenge by demonstrating measurements of molecular diffusivity of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. The approach, termed single-molecule microfluidic diffusional sizing (smMDS), allows individual molecules to be counted directly, that is, in a digital manner, to enable calibration-free single-molecule diffusional-sizing-based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures. Applying smMDS to a variety of protein systems, we show that the high sensitivity provided by smMDS enables ultrasensitive sizing of proteins down to the femtomolar concentration range. We further demonstrate the applicability of the approach towards affinity profiling of protein interactions at the single-molecule level and illustrate the potential of smMDS in resolving different assembly states of high- and low-molecular weight protein oligomers. Furthermore, we highlight the digital nature of the detection process by sizing multiple protein species within complex aggregation mixtures. Finally, we apply the approach to characterize nanoscale clusters of a phase separating protein system. Taken together, smMDS constitutes a versatile approach for digital, in-solution characterization of the sizes, interactions, and assembly states of proteins. We anticipate that smMDS will facilitate the discovery of new biomolecular mechanisms of proteins and will find broad applicability in the analysis of protein complexes in the biological, biophysical, and biomedical sciences, and beyond.

biophysics↗

bioRxiv2023

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce temporal resolution by requiring lower labelling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMFLM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We then experimentally demonstrate the high accuracy (>99.2 {+/-} 0.1%, 0.1 locs m-2) and sensitivity (>86.6 {+/-} 0.9%, 0.1 locs m-2) of SMLFM at point detection through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high density volumetric imaging (0.15 locs m-2) in dense cytosolic tubulin datasets.

biophysics↗

bioRxiv2023

Exhaustive benchmarking of de novo assembly methods for eukaryotic genomes

The assembly of reference-quality, chromosome-resolution genomes for both model and novel eukaryotic organisms is an increasingly achievable task for single research teams. However, the overwhelming abundance of sequencing technologies, assembly algorithms, and post-assembly processing tools currently available means that there is no clear consensus on a best-practice computational protocol for eukaryotic de novo genome assembly. Here, we provide a comprehensive benchmark of 28 state-of-the-art assembly and polishing packages, in various combinations, when assembling two eukaryotic genomes using both next-generation (Illumina HiSeq) and third-generation (Oxford Nanopore and PacBio CLR) sequencing data, at both controlled and open levels of sequencing coverage. Recommendations are made for the most effective tools for each sequencing technology and the best performing combinations of methods, evaluated against common assessment metrics such as contiguity, computational performance, gene completeness, and reference reconstruction, across both organisms and across sequencing coverage depth.

bioinformatics↗

bioRxiv2023

Pyro: A Comprehensive Pipeline for Eukaryotic Genome Assembly

1The assembly of reference-quality, chromosome-level genomes for both model and novel eukaryotic organisms is an increasingly achievable task for single research teams. However, the broad variety of sequencing technologies, assembly algorithms, and post-assembly processing tools currently available means that there is no clear consensus on a best-practice computational protocol for eukaryotic de novo genome assembly. An ever-increasing field of algorithms and packages with unique parameters, setup requirements, and environments makes it difficult for groups to pick up and test new tools, despite potential benefits. Here, we present a comprehensive Snakemake-based pipeline for eukaryotic genome assembly, Pyro, to further assist future de novo assembly and benchmarking projects. Pyro combines 20 assembly and eight polishing packages, comprising 30 different assembly approaches and up to 48 different polishing approaches in combination. These are available across Illumina short-read, Nanopore and PacBio CLR long-read technologies in one container, complete with data preparation, quality metric calculation and result reporting. We demonstrate Pyro effectiveness by running Pyro on publicly available Illumina, Nanopore and PacBio CLR read sets for Arabidopsis thaliana, producing 12 candidate assembly options with minimal initial input or configuration, each with extremely high contiguity and completeness. Pyro is highly customizable to expert needs, while also providing an accessible suggested set of tools for more casual users based on simple inputs. Pyro is available as a Singularity container suitable for execution on any Unix-compatible OS, and is freely available on GitHub (https://github.com/genomeassembler/pyro). This pipeline provides a one-stop solution for a variety of de novo eukaryotic genome assembly needs, and will also assist in the assessment of new tools as a convenient benchmark-generating platform.

bioinformatics↗

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↗

bioRxiv2022

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a reservoir of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

biophysics↗