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Introduction
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Publications (68)
Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods availab...
Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the da...
Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods availab...
Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualizing information derived from microscopy imaging data of biological samples. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of...
In recent years, the development of analytical approaches to super-resolution microscopy has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit wit...
Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualizing information derived from biological microscopy imaging data. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of the number...
In recent years, the development of analytical approaches to super-resolution microscopy has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit wit...
Significance
Cell division is an essential requirement for life. Division requires mechanical forces, often exerted by protein assemblies from the cell interior, that split a single cell into two. Using coarse-grained computer simulations and live cell imaging we define a distinct cell division mechanism—based on the forces generated by the superco...
Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by protein assemblies. Here we developed the first physical model for the division of archaeal cells, which despite their structural simplicity share machinery and evolutionary orig...
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an ‘open mitosis’ or by reshaping an intact nucleus and then dividing it into two in a ‘closed mitosis’2,3. Mitosis has been stud...
Proteasomal control of division in Archaea
In eukaryotes, proteasome-mediated degradation of cell cycle factors triggers mitotic exit, DNA segregation, and cytokinesis, a process that culminates in abscission dependent on the protein ESCRT-III. By studying cell division in an archaeal relative of eukaryotes, Tarrason Risa et al. identified a role f...
Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. Although similar techniques have been applied to the study of halophilic archaea [1, 2, 3, 4, 5], our ability to explore the cell biology of thermophilic archaea has been limited by the technical challenges of imaging at high temperature...
Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we...
Super-Resolution Microscopy enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for l...
At the end of mitosis, eukaryotic cells must segregate both copies of their replicated genome into two new nuclear compartments (1). They do this either by first dismantling and later reassembling the nuclear envelope in a so called “open mitosis”, or by reshaping an intact nucleus and then dividing into two in a “closed mitosis” (2, 3). However, w...
The archaeon Sulfolobus acidocaldarius is a relative of eukaryotes known to progress orderly through its cell division cycle despite lacking obvious CDK/cyclin homologues. Here, in exploring the mechanisms underpinning archaeal cell division cycle control, we show that the proteasome of S. acidocaldarius, like its eukaryotic counterpart, regulates...
Super-Resolution Microscopy enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for l...
Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability...
Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability...
Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows. Here, we present a robust, open-source approach for treating, labelling and imaging live or fixed cells in automated sequences. NanoJ-Fluidics is based on low-cost Lego hardware controlled by ImageJ-based software, making hig...
Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM d...
Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging d...
Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resol...
Fluorescence microscopy is a key driver of discoveries in the life-sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging d...
Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary informatio...
Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide pale...
Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolu...
The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise...
Super-Resolution Radial Fluctuations (SRRF) is a technique for achieving super-resolution in samples that are typically challenging for SMLM, such as live cells and samples labelled with conventional non-blinking fluorophores. SRRF was first released as a GPU-enabled open source ImageJ plugin for post-processing of acquired time series [1]; however...
Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, th...
Super-resolution radial fluctuations (SRRF) is a synthesis of temporal fluctuation analysis and localization microscopy [1]. One of the key differences between SRRF and other super-resolution methods is its applicability to live-cell dynamics because it functions across a very wide range of fluorophore densities and excitation powers. SRRF can be a...
Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challe...
Live-cell microtubule dynamics imaged with SRRF. EGFP-labeled microtubules in live HeLa cells imaged at 100 frames per second, yielding 1 super resolution frame per second. First half: top panel TIRF imaging, bottom panel SRRF rendering, inset corresponds to zoom for second half of the movie. Second half: expansions of inset, plot shows the normali...
Long-term SRRF imaging of microtubule dynamics. EGFP-labeled microtubules in live HeLa cells imaged at 100 frames per second, yielding 1 super resolution frame per second, every 25 minutes for 8 hours. Top panel: TIRF imaging, bottom panel: SRRF rendering. Between 5 and 6 hours the cell lifts from the coverslip, undergoing mitosis. Scale bar is 5 μ...
T cell spreading following drop imaged with SRRF. LifeAct-GFP-labeled actin in a live Jurkat T cell imaged at 100 frames per second, yielding 1 super resolution frame per second. The T cell is dropped onto an anti-CD3 coated coverslip, leading to spreading of the cell and reorganization of the intracellular actin network. Left panel: TIRF movie, ri...
The SRRF algorithm. The SRRF algorithm source code and plugin for ImageJ and Fiji are provided along with a manual providing instructions for installation and use.
Rendering of the radiality transform for various parameters and input point-spread-functions. The radiality transform is shown for the real image (i.e. diffraction-limited image) under a wide range of conditions. In the first half of the movie the radiality transform of an image of a single fluorophore is considered. Firstly, the ring radius over w...
Mitochondrial dynamics imaged with SRRF. Mitotracker Red-labeled mitochondria in live HeLa cells imaged at 100 frames per second, yielding 1 super resolution frame per second. Top panel: TIRF movie, bottom panel: SRRF rendering. Scale bar is 5 μm.
Supplementary Figures 1-12, Supplementary Tables 1- 2, Supplementary Notes 1-3, Supplementary Methods, Supplementary References.
Non-directed actin rearrangement following stimulation with anti-CD28 imaged with SRRF. LifeAct-GFP-labeled actin in a live Jurkat T cell imaged at 100 frames per second, yielding 1 super resolution frame per second. An anti-CD28 coated coverslip is used to stimulate immunological synapse formation. Left panel: TIRF movie, right panel: SRRF renderi...
Retrograde flow of actin during immunological synapse formation upon stimulation with anti-CD3 imaged with SRRF. LifeAct-GFP-labeled actin in a live Jurkat T cell imaged at 100 frames per second, yielding 1 super resolution frame per second. An anti-CD3 coated coverslip is used to stimulate immunological synapse formation. Left panel: TIRF movie, r...
Retrograde flow of actin during immunological synapse formation upon stimulation with anti-CD3&CD28 imaged with SRRF. LifeAct-GFP-labeled actin in a live Jurkat T cell imaged at 100 frames per second, yielding 1 super resolution frame per second. An anti-CD3&CD28 coated coverslip is used to stimulate immunological synapse formation. Left panel: TIR...
The ability to accurately and reliably quantify viral infection is essential to basic and translational virology research. Here, we describe a simple and robust automated method for using fluorescence microscopy to estimate the proportion of virally infected cells in a monolayer. We provide details of the automated analysis workflow along with a fr...
Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, w...
Many biological structures exist on a scale smaller than can be resolved by conventional fluorescence microscopy, which has limited the study of cellular processes. For this reason, there has been a large amount of research over the past decade dedicated to the development of super resolution microscopy techniques, which allow optical imaging of st...
We demonstrate a new method for obtaining sub-diffraction resolution in fluorescence microscopy. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured (fundamental Gaussian) continuous wave stimulated emission depletion. A reduced point spread functions (PSF) is obtained by the rec...
The information sent from each cochlear inner hair cell (IHC) to the afferent nerve is determined by 10-20 ribbon synapses, structures specialised for rapid release of vesicles upon cell depolarization. To study the IHC calcium domains during transmitter release in mature wild-type mice, we have imaged and simultaneously measured currents in IHCs t...
