Biology subjects

Gregor, I.

Publications and source records attributed to Gregor, I..

9 recordsLinked to original sources

bioRxiv2026

Universal approach to wave-optical calculations of point spread functions in microscopy (and beyond)

Optical microscopy is fundamental to modern life-science research, yet interpreting its results requires precise modelling of point spread functions (PSFs) within complex environments. This manuscript introduces a versatile and efficient approach to wave-optical PSF calculations that extends existing frameworks by incorporating detection PSF modelling through the principle of reciprocity. Accompanying this work is a free MATLAB software package centred on a single, minimalistic core function, PlaneWaveExc.m, which utilizes a plane-wave superposition based on the Richards-Wolf model. Despite its simplicity, the framework accounts for "real-life" complexities such as systemic aberrations, arbitrary amplitude and phase modulations, and stratified media with complex-valued refractive indices. We demonstrate the softwares broad applicability through diverse case studies, including single-molecule imaging, STED microscopy, the segmented aperture of the James Webb Space Telescope, and coherent wide-field iSCAT microscopy. Each example is supported by dedicated scripts to facilitate adaptation for specific research needs.

biophysics↗

bioRxiv2023

Doubling the resolution of single-molecule localization microscopy with image scanning microscopy

In this study, we integrate a single-photon detector array into a Confocal Laser Scanning Microscope (CLSM), enabling the combination of Fluorescence Life-time Single-Molecule Localization Microscopy (FL-SMLM) with Image Scanning Microscopy (ISM). This unique combination delivers a two-fold improvement in lateral localization accuracy for SMLM while maintaining its simplicity. More-over, the addition of lifetime information from our CLSM eliminates chromatic aberration, particularly crucial for achieving few-nanometer resolution in SMLM. Our approach, named Fluorescence Lifetime ISM-SMLM (FL-iSMLM), is demon-strated through direct Stochastic Optical Reconstruction Microscopy (dSTORM) and DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) experiments on fluorescently labelled cells, showcasing both resolution enhancement and fluorescence lifetime multiplexing capabilities.

biophysics↗

bioRxiv2022

Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins

Metal-Induced Energy Transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and sub-cellular components across multiple timescales, from a few milliseconds to hours, with negligible phototoxic effects.

biophysics↗

bioRxiv2022

Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass transition temperature hampers efficient photoswitching at low temperature. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent protein at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at [~]110 K. At this cryogenic temperature, on-off photoswitching involves the formation of 2 dark states with blue shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these dark states can be switched back to the fluorescent state by 405 nm light, while both of them are sensitive to UV light at 355 nm. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. It suggests that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 or possibly other FPs could improve the achievable effective labeling efficiency in this technique. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/504779v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1d5ed02org.highwire.dtl.DTLVardef@1bb2359org.highwire.dtl.DTLVardef@10dc6b1org.highwire.dtl.DTLVardef@16fcd94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Instant three color multi-plane fluorescence microscopy

One of the most widely used microscopy techniques in biology and medicine is fluorescence microscopy, offering high specificity in labeling as well as maximum sensitivity. For live cell imaging, the ideal fluorescence microscope should offer high spatial resolution, fast image acquisition, three-dimensional sectioning, and multi-color detection. However, most existing fluorescence microscopes have to compromise between these different requirements. Here, we present a multi-plane multi-color wide-field microscope that uses a dedicated beam-splitter for recording volumetric data in eight focal planes and for three emission colors with frame rates of hundreds of volumes per second. We demonstrate the efficiency and performance of our system by three-dimensional imaging of multiply labeled fixed and living cells.

biophysics↗

bioRxiv2020

Three-dimensional beating dynamics of Chlamydomonas flagella

Axonemes are the basic structure of motile cilia and flagella, and the investigation of how they function and move requires rapid three-dimensional imaging. We built a multi-plane phase-contrast microscope for imaging the three-dimensional motion of unlabeled flagella of the model organism Chlamydomonas reinhardtii with sub-m spatial and 4 ms temporal resolution. This allows us to observe not only bending but also the three-dimensional torsional dynamics of these small structures. We observe that flagella swim counter-clockwise close to a surface, with negatively-valued torsion at their basal and positively-valued torsion at their distal tips. To explain the torsional dynamics and signature, we suggest the existence of an intrinsic negative twist at the basal end that is untwisted by active positive-twist-inducing dynein motor proteins. Moreover, dyneins walking towards the basal induce an opposite twist at the distal tip. Bending of the whole axoneme structure then translates this twist into an observable torsion. This interconnection between chiral structure, twist, curvature, and torsion is fundamental for understanding flagellar mechanics.

biophysics↗

bioRxiv2018

Photon yield enhancement of red fluorophores at cryogenic temperatures

Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 {middle dot} 106 to 11 {middle dot} 106 photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

biophysics↗

bioRxiv2017

Protein phosphatase V ensures timely cell cycle remodeling during the mid-blastula transition in Drosophila

Cell cycle remodeling from fast nuclear cycles to a generic cell cycle mode is a major feature of the mid-blastula transition (MBT) in Drosophila. Remodeling occurs when Twine/Cdc25 falls below a critical threshold. Timing is based on Twine destabilization induced by zygotic transcription. It is conceivable that appropriate starting levels are also important for timely reaching the threshold. Mechanisms for controlling Twine levels at the onset of MBT are unknown. Here we identify a function of the protein phosphatase V in this mechanism. Twine was increased in PpV mutants, whereas the decay rate was comparable to wildtype. PpV mutants frequently underwent an extra nuclear division. We detected PpV-dependent phosphosites in Twine. Phosphosite mutants contain higher Twine levels and frequently underwent an extra nuclear division, comparable to PpV mutants. Our data support a model that the cell cycle remodeling is controlled by induced destabilization and PpV-d ependent control of Twine levels.

developmental biology↗

bioRxiv2017

Critical Assessment of Metagenome Interpretation - a benchmark of computational metagenomics software

In metagenome analysis, computational methods for assembly, taxonomic profiling and binning are key components facilitating downstream biological data interpretation. However, a lack of consensus about benchmarking datasets and evaluation metrics complicates proper performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on datasets of unprecedented complexity and realism. Benchmark metagenomes were generated from ~700 newly sequenced microorganisms and ~600 novel viruses and plasmids, including genomes with varying degrees of relatedness to each other and to publicly available ones and representing common experimental setups. Across all datasets, assembly and genome binning programs performed well for species represented by individual genomes, while performance was substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below the family level. Parameter settings substantially impacted performances, underscoring the importance of program reproducibility. While highlighting current challenges in computational metagenomics, the CAMI results provide a roadmap for software selection to answer specific research questions.

bioinformatics↗