Biology subjects

Baddeley, D.

Publications and source records attributed to Baddeley, D..

13 recordsLinked to original sources

bioRxiv2025

A correlative quantitative phase contrast and fluorescence super-resolution microscope for imaging molecules in their cellular context

Fluorescence microscopy has been widely used to reveal the spatial distribution of specifically labeled molecules, but it is blind to cellular context. Quantitative phase contrast microscopy (QPC) provides such complementary information. Here we have developed a platform that combines the QPC technique of correlative orientation-independent differential interference contrast (OI-DIC) microscopy with single-molecule super-resolution fluorescence microscopy. We demonstrate a detection sensitivity of 0.05 nm optical path difference, sufficient to detect single microtubules, and show its capability of 3D super-resolution fluorescence imaging in the cellular context. Additionally, we report deep-learning enabled digital staining, identifying nuclei, mitochondria and lipid droplets from OI-DIC data and demonstrate the potential of this approach for long-term live-cell imaging of organelles of interest without the need for fluorescence. OI-DIC can be easily integrated into most fluorescence microscopes and is readily adoptable by microscopy labs. ONE-SENTENCE TEASERA highly sensitive technique to visualize sub-cellular structures, dynamics, and molecules in their cellular context.

cell biology↗

bioRxiv2023

The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ

The endoplasmic reticulums (ER) structure is directly linked to the many functions of the ER but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear-shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4. SummaryRtn4 forms linear-shaped oligomers that contain an average of five Rtn4 proteins, localize to the sides of elliptical tubules, prefer orientations near parallel to the tubule axis, and increase local curvature of the ER membrane by increasing local Rtn4 density.

cell biology↗

bioRxiv2023

Extracting nanoscale morphology from localizations

Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that is manually cleaned and curated. Here we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy (SMLM) data. This makes it possible to study the shape of specifically-labelled membraneous structures inside of cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on SMLM data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment. SIGNIFICANCEWe introduce a novel tool for reconstruction of subcellular membrane surfaces from single-molecule localization microscopy data and use it to visualize and quantify local shape and membrane-membrane interactions. We benchmark its performance on simulated data and demonstrate its fidelity to experimental data.

biophysics↗

bioRxiv2020

PYMEVisualize: an open-source tool for exploring 3D super-resolution data

Localization-based super-resolution microscopy techniques such as PALM, STORM, and PAINT are increasingly critical tools for biological discovery. These methods generate lists of single fluorophore positions that capture nanoscale structural details of subcellular organisation, but to develop biological insight, we must post-process and visualize this data in a meaningful way. A large number of algorithms have been developed for localization post-processing, transforming point data into representations which approximate traditional microscopy images, and performing specific quantitative analysis directly on points. Implementations of these algorithms typically stand in isolation, necessitating complex workflows involving multiple different software packages. Here we present PYMEVisualize, an open-source tool for the interactive exploration and analysis of 3D, multicolor, single-molecule localization data. PYMEVisualize brings together a broad range of the most commonly used post-processing, density mapping, and direct quantification tools in an easy-to-use and extensible package. This software is one component of the PYthon Microscopy Environment (python-microscopy.org), an integrated application suite for light microscopy acquisition, data storage, visualization, and analysis built on top of the scientific Python environment.

biophysics↗

bioRxiv2020

Fluorogenic probe for fast 3D whole-cell DNA-PAINT

DNA-PAINT is an increasingly popular super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and very slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. We present a fluorogenic DNA-PAINT probe that solves these problems and demonstrate 3D imaging without the need for optical sectioning and a 26-fold increase in imaging speed over regular DNA-PAINT.

biophysics↗

bioRxiv2019

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time-and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

biophysics↗

bioRxiv2019

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Actin is required for clathrin-mediated endocytosis (CME) in yeast. Experimental observations indicate that this actin assembly generates force to deform the membrane and overcome the cells high turgor pressure, but the precise molecular details remain unresolved. Based on previous models, we predicted that actin at endocytic sites continually polymerize and disassemble, turning over multiple times during an endocytic event. Here we applied single-molecule speckle tracking in live fission yeast to directly measure this predicted turnover within the CME assembly for the first time. In contrast with the overall ~20-sec lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 sec, and high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins motions. These results indicate that rapid and continuous turnover is a key feature of the endocytic machinery and suggest revising quantitative models of force production.

cell biology↗

bioRxiv2019

Nanoscale subcellular architecture revealed by multicolor 3D salvaged fluorescence imaging

Combining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.\n\nOne Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

biophysics↗

bioRxiv2019

3D Multicolor Nanoscopy at 10,000 Cells a Day

Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and are invaluable to investigate sub-cellular organization. The multitude of camera frames required to reconstruct a super-resolved image limits the typical throughput of these techniques to tens of cells per day, rendering these methods incompatible with large-scale cell biological or clinical application. STORM acquisition rates can be increased by over an order of magnitude, however the data volumes of about 40 TB a day and concomitant analysis burdens exceed the capacity of existing workflows. Here we present an integrated platform which transforms SMLM from a trick-pony technique into a work horse for cell biology. We leverage our developments in microscopy-specific data compression, distributed storage, and distributed analysis to automatically perform real-time localization analysis, which enable SMLM at throughputs of 10,000 cells a day. We implemented these advances in a fully-integrated environment that supports a highly-flexible architecture for distributed analysis, enabling quickly- and graphically-reconfigurable analyses to be automatically initiated from the microscope during acquisition, remotely executed, and even feedback and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, the PYthon-Microscopy Environment (PYME), is easily configurable for hardware control on custom microscopes, and includes a plugin framework so users can readily extend all components of their imaging, visualization, and analysis pipeline. PYME is cross-platform, open source, and efficiently puts high-caliber visualization and analysis tools into the hands of both microscope developers and users.

biophysics↗

bioRxiv2018

A stable, high refractive index, switching buffer for super-resolution imaging

dSTORM super-resolution imaging relies on switching buffers to enable dye molecules to enter and exit a metastable dark state. Current buffers have a very limited shelf life of approximately 1 day and poorly match sample refractive index, impacting negatively on measurement reproducibility and image fidelity. We present a buffer based on chemical, rather than enzymatic, oxygen scavenging which exhibits dramatically improved stability, switching speed, contrast, and index matching.

biophysics↗

bioRxiv2017

Nano-scale size holes in ER sheets provide an alternative to tubules for highly-curved membranes

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Super-resolution microscopy recently revealed densely packed, rapidly moving ER tubules, highlighting the importance of revisiting classical views of ER structure with high spatial resolution in living cells. Using live-cell Stimulated Emission Depletion (STED) microscopy, we show highly dynamic, subdiffraction-sized holes in ER sheets. Holes coexist with uniform sheet regions and are distinct from tubular ER structures. The curvature-stabilizing reticulon protein Rtn4 localizes to these holes and the ER luminal tether Climp63 controls their diameter and mobility. Analytical modeling demonstrates that holes in ER sheets can serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction, thus providing an explanation for how the ER locally alters its morphology on fast time-scales.\n\nOne Sentence SummaryDynamic nano-scale sized holes are prominent features of ER sheets that serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction.

cell biology↗

bioRxiv2017

Simultaneously measuring image features and resolution in live-cell STED images

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a non-biological proxy for a point-like object, such as a fluorescent bead. While appropriate for confocal microscopy, bead-based measurements are problematic for Stimulated Emission Depletion (STED) and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. We demonstrate that for a known geometry, e.g. tubules, the resolution can be accurately measured by fitting a model that accounts for both the Point Spread Function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique, Nested-loop Ensemble PSF (NEP) fitting. NEP fitting enables extraction of the size of cellular features and the PSF in fixed-cell and live-cell images without relying on beads or pre-calibration. We validate our technique using fixed microtubules and apply it to measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. NEP fitting has been implemented as a plugin for the PYthon Microscopy Environment (PYME), a freely available and open source software.

cell biology↗

bioRxiv2016

New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics

Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. We have developed a novel, broadly applicable, fluorescent labeling and imaging protocol, called Single-molecule Recovery After Photobleaching (SRAP), which allowed us to reveal the heterogeneous dynamics of the eisosome, a multi-protein structure on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the core eisosome BAR domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, recorded the binding of individual Pil1p molecules with ~20 nm precision. Further analysis of these dynamic structures and comparison to computer simulations allowed us to show that Pil1p exchange is spatially heterogeneous, supporting a new model of the eisosome as a dynamic filament.\n\nAbbreviations used

cell biology↗