Biology subjects

Soeller, C.

Publications and source records attributed to Soeller, C..

13 recordsLinked to original sources

bioRxiv2026

Characterizing MINFLUX imaging performance with DNA origami

MINFLUX, a second-generation super-resolution technique, can localize fluorescent markers approaching single-nanometer precision in three dimensions. Similar to previous super-resolution methodologies, the extended duration of acquisitions can result in drift that needs accurate correction to enable proper analysis and interpretation of the collected data. Here, we use DNA origami, housing sites of known spatial distribution fitted with repeat-domain docking strands for DNA-PAINT imaging to characterize imaging performance over extended duration MINFLUX acquisitions (6-20 h). Repeat-domain docking strands overcome site-loss and reveal residual drift in prolonged MINFLUX 3D acquisitions that we correct with an algorithm exploiting time-correlated shifts of localizations around identified DNA origami sites. Following correction of residual drift the site precision, i.e. the scatter of localizations around sites, is [~]2 nm in all directions. Comparison of site precision from extended repeat-domain docking strands with site precision from standard short 8-10 nucleotide docking strands exhibits no detectable loss of site precision. By adding DNA origami structures to mounted biological samples we apply our approach to the imaging of the cardiac ryanodine receptor 2 in cryosectioned heart tissue. The data suggests that for these protein targets single domain markers with repeat domain docking strands may be directly used for residual drift correction, simplifying sample preparation and acquisition protocols.

biophysics↗

bioRxiv2025

Optimizing effective labeling efficiency in MINFLUX 3D DNA-PAINT microscopy by maximizing marker detection probability.

MINFLUX is a powerful single-molecule approach capable of achieving high spatially isotropic resolution in three dimensions. Current implementations collect localizations strictly serially, but criteria for when to terminate acquisition are often unclear. We therefore systematically investigate the time course of effective labeling efficiency (ELE) and achievable saturation values in MINFLUX 3D DNA-PAINT microscopy of Nup96 proteins in a U-2 OS-Nup96-mEGFP modified cell line using a commercial MINFLUX microscope. ELE was measured with a maximum-likelihood template fitting assisted quantitative procedure. We collected data measured over various scan sizes and achieved ELE values of [~]60% after passing a time interval dependent on the region size, typically requiring long-duration acquisitions over several hours. Our data and a simple model suggest that maximizing marker detection is key to achieving the limits set by chemical labeling efficiency. A factor limiting the marker detection probability when using conventional DNA-PAINT markers is docking strand site-loss, observed over the duration required to build up the image data of MINFLUX acquisitions, which also limits the achievable number of labeling site visits to values around 1-3. Using repeat DNA-PAINT, i.e. employing oligonucleotide sequences with repeated docking sites, we observed greatly reduced site-loss and could increase the number of individual visits to site locations by more than threefold over the same period. Additionally, this enabled increasing stringency criteria for labeling (i.e. higher threshold values) and maximizing marker detection probabilities so that ELE reaches the limits set by chemical labeling efficiency.

biophysics↗

bioRxiv2024

Reduced non-specific binding of super-resolution DNA-PAINT markers by Shielded DNA-PAINT labeling protocols

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events [~]5 fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific background.

biophysics↗

bioRxiv2023

Analysis of RyR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D MINFLUX microscopy

The cardiac type 2 ryanodine receptor (RyR2) is a large homotetramer of a [~]560 kD subunit and is the molecular pathway through which the majority of Ca2+ enters the cytosol during cardiac activation. It constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative giving rise to local release events termed Ca2+ sparks. Accordingly, the molecular distribution of RyR2 in cardiac myocytes has been of great interest. Here we present the first purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX fluorescence super-resolution microscopy. We demonstrate that by using single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions we can determine the location of individual RyR2 subunits with high precision ([~]3 nm) in all directions. Combining MINFLUX with DNA-PAINT, to maximize detection efficiency, we measured in situ labeling efficiencies using NPC structures as reference and regularly achieved efficiencies around 50%, which would translate to RyR2 detection efficiencies close to 95%, i.e. the probability that at least one subunit is detected, if target accessibility is similar. Using this approach, we detect dense and extended RyR2 expression in HEK cells with some clusters spanning several micrometres in extent and containing several hundred RyR2s. Similarly, myocytes from PA-RFP RyR2 mice contained large clusters containing many tens of RyR2s. The new data also suggests a resolution to apparent discrepancies between previous data from electron microscopy and super-resolution data. The methodology developed here will be critical to reveal the full complexity of RyR2 and related Ca2+ handling proteins in 3D as well as their relationship to contractile function. Our new approaches should be applicable to other multi-subunit complexes in cardiac muscle and other cell types.

physiology↗

bioRxiv2022

Prolonged β-Adrenergic Stimulation Disperses Ryanodine Receptor Clusters in Cardiomyocytes: Implications for Heart Failure

Ryanodine Receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that "dispersion" of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged {beta}-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion, and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hour triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of CaMKII reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of {beta}-adrenergic stimulation over the longer term, and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology. Significance statementThe heartbeat is triggered by the release of Ca2+ from Ryanodine Receptors (RyRs) within cardiomyocytes. Recent data indicate RyR arrangement is highly malleable. However, mechanisms controlling RyR reorganisation and the subsequent impact on Ca2+ homeostasis remain unclear. Here, we show that prolonged {beta}-adrenergic stimulation causes RyR clusters to disperse, drastically altering the frequency and kinetics of Ca2+ release events called "Ca2+ sparks" in a process that is dependent on CaMKII and PKA. In healthy cells, these compensatory effects protect against arrhythmogenic Ca2+ over-activity. However, during heart failure, RyR hyper-phosphorylation and dispersion impairs Ca2+ release and cardiac performance. Thus, RyR localization and function are intimately linked via channel phosphorylation which, while finely tuned in health, underlies impaired cardiac function during pathology.

cell biology↗

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

Ca2+ release via IP3Rs increases RyR mediated Ca2+ spark frequency in ventricular cardiomyocytes without altering spark amplitude and duration

Calcium (Ca2+) plays a critical role in the excitation contraction coupling (ECC) process that governs the contraction of cardiomyocytes during each heartbeat. While ryanodine receptors (RyRs) are the primary Ca2+ channels responsible for mediating cell-wide Ca2+ transients during ECC, Ca2+ release via inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) have been reported to elicit ECC-modulating effects. Recent studies suggest that the proximal localization of IP3Rs at dyads grants their ability to modify the occurrence of Ca2+ sparks (elementary Ca2+ release events that constitute ECC-associated Ca2+ transients) which may underlie the modulatory effects on ECC. Here, we aim to uncover the mechanism by which IP3Rs affect Ca2+ spark dynamics. To this end, we developed a mathematical model of the dyad that incorporates IP3Rs to reveal their impact on local Ca2+ handling and corresponding Ca2+ spark formation. Consistent with published experimental data, our model predicts that the propensity for Ca2+ spark formation increases with IP3R activity. Our simulations support the hypothesis that IP3R activity elevates Ca2+ within the dyad, sensitizing proximal RyRs for future release. However, this lowers Ca2+ in the JSR available for release and thus results in Ca2+ sparks with the same duration but lower amplitudes.

biophysics↗

bioRxiv2020

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA imagers to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.

molecular biology↗

bioRxiv2019

In vitro reconstitution of branched microtubule nucleation

Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator,{gamma} -Tubulin Ring Complex ({gamma}-TuRC)1-4, within the cell. A conserved MT-associated protein complex, Augmin, recruits{gamma} -TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed \"branched\" MT nucleation5-9. Here, we purify endogenous, GFP-tagged Augmin and{gamma} -TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates{gamma} -TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-{gamma}-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branched MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

cell biology↗

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

Detecting nanoscale distribution of protein pairs by proximity-dependent super-resolution microscopy

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in-situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present Proximity-Dependent PAINT (PD-PAINT), a method for sub-diffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA labels anchored to the target species. Labeled protein pairs are then imaged with high contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution and the ability to image quantitatively.

molecular biology↗

bioRxiv2019

Detecting RyR clusters with CaCLEAN: influence of spatial distribution and structural heterogeneity

In cardiomyocytes, the coordinated release of calcium ions from intracellular stores through ryanodine receptor (RyR) clusters is key to the generation of a calcium transient and induction of contraction. Recently, a deconvolution algorithm from radio astronomy was adapted and applied to analysis of calcium fluorescence imaging of cardiomyocytes. The algorithm, CaCLEAN, showed potential in revealing both the spatial locations of RyR clusters and their functional response in living cells. However, whether the RyR clusters identified by CaCLEAN analysis of the imaging data were true or false positives remained unvalidated in the absence of ground truth values. In this work, a structurally realistic finite element model was developed to simulate reaction-diffusion of calcium emanating from RyR clusters during the rising phase (first 30 ms) of the calcium transient. The effect of two sets of factors were examined with the model: (1) the number and spacing of simulated RyR clusters and (2) the effect of mitochondria acting as barriers to diffusion. Confocal fluorescence microscopy images were simulated from the model results and analysed using CaCLEAN. The performance of CaCLEAN was found to be sensitive to cluster spacing and distance from the imaging plane. In a case with sparsely-packed clusters, detection recall and precision were 0.82 for clusters up to 610 nm from the imaging plane; in a densely-packed cluster case, recall and precision were 0.69 for clusters up to 280 nm from the imaging plane. Users interested in applying CaCLEAN to their data should therefore consider the likely density of cluster distributions and the trade-off between precision and recall when determining the maximum relevant depth of RyR clusters in their application.

bioengineering↗

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↗