Biology subjects

Thiele, J. C.

Publications and source records attributed to Thiele, J. C..

10 recordsLinked to original sources

bioRxiv2026

Mass-specific single molecule pull-down from complex mixtures with bilayer-assisted mass photometry

Determining the identity, quantity and interactions of proteins is fundamental to studies of biomolecular function and (dis)regulation in disease. Here, we combine mass photometry with supported lipid bilayers (SLBs) to specifically recruit proteins of interest from complex mixtures such as cell lysate and demonstrate sensitive detection and mass measurement of single biomolecules and their complexes without prior purification. Extending to heterologously expressed proteins, we provide rapid information on oligomerisation and concentration in lysate. Introduction of polyethylene glycol in the SLBs passivates against a variety of cell lysates and serum, allows specific recruitment of proteins from complex mixtures and mass-specific detection and affinity measurement at sub-nM concentrations. Our approach preserves the mass resolution and accuracy of mass photometry, thereby enabling visualisation and quantification of oligomeric states, interactions and complexes directly from cell lysates.

biophysics↗

bioRxiv2026

Modular DNA nanosensors enable state-aware multiplexed single-molecule mass photometry in complex media

Proteins exist in diverse biochemical states, including oligomers, complexes, heterogeneous proteoforms and shed fragments that encode functional and regulatory information. Yet, these molecular states remain difficult to resolve with existing analytical techniques, which typically require labelling, immobilisation or amplification and collapse this information into a single readout. Here, we introduce a modular platform that integrates programmable DNA nanostructures with single-molecule mass photometry for rapid, label-free and multiplexed protein analysis in native and complex media, without washing, enrichment or immobilisation, making native biochemical states observable directly in serum and plasma. DNA nanostructures act as nanosensors whose mass and mobility on supported lipid bilayers provide orthogonal identifiers for target identity and biochemical state, thereby decoupling recognition from readout. We define the analytical specificity and response window, demonstrate quantitative affinity determination, and resolve oligomeric and proteoform differences under native conditions. The nanosensors are rapidly reprogrammable to new targets, support multiplexed detection with internal controls for non-specific interactions, and enable selective resetting via strand displacement. Together, these capabilities establish nanoscale programmability as a route to state-resolved single-molecule protein profiling adaptable to both diagnostic and mechanistic applications.

biophysics↗

bioRxiv2026

Continuous mass photometry by single molecule trapping

The dynamic choreography of biomolecular interactions underpins the processes of life, but its direct observation remains challenging. Here, we introduce confined diffusion mass photometry, enabling hour-long, mass-resolved observation of individual biomolecules, their complexes and interactions with up to sub-kDa mass precision and ms temporal resolution. Our approach represents a quantitative time-resolved single-molecule measurement modality for studying complex biomolecular mechanisms in action.

biophysics↗

bioRxiv2025

Deep learning-based event classification of mass photometry data for optimal mass measurement at the single-molecule level

Mass photometry (MP) is a powerful technique for studying biomolecular structure, interactions, and dynamics in solution. It detects and quantifies small reflectivity changes at a glass-water interface during protein (un)binding, with signals typically averaged over 100 milliseconds. However, particle motion at the point of single-molecule measurement can compromise key metrics such as mass resolution, sensitivity, and concentration. We present a three-dimensional convolutional residual network trained via supervised learning to classify landing events based on their spatiotemporal dynamics. By analysing 3D event thumbnails, our method isolates optimal single-molecule measurements, eliminating cumulative histogram artifacts and improving resolving power by up to a factor of 2. Validated across diverse experimental datasets--including resolved and partially resolved samples, and varying masses, concentrations, and integration times--our approach delivers robust performance under (sub)optimal conditions. Our approach provides measurement-level data-driven feedback, facilitating high quality MP measurements in challenging scenarios.

biophysics↗

bioRxiv2024

Best practice mass photometry: A guide to optimal single molecule mass measurement

Mass photometry (MP) has emerged as a powerful approach to study biomolecular structure, dynamics and interactions. The capabilities of the method ultimately hinge on the ability to accurately measure the tiny optical contrast generated by individual molecules landing at a glass-water interface, which enables mass-resolved quantification of biomolecular mixtures. Ideally, this capability is only limited by shot noise inherent to photon detection, but in practice depends on additional parameters and details of the assay. Here, we focus on the key parameters affecting MP performance, and present simple steps that can be taken to achieve optimal MP performance in terms of mass resolution, quantitative detection limit and analyte concentration range without compromising the ease and simplicity of the technique.

biophysics↗

bioRxiv2024

Three-dimensional multi-target super-resolution microscopy of cells using Metal-Induced Energy Transfer and DNA-PAINT

AbsrtactAchieving nanometer precision in 3D remains a major challenge in super-resolution microscopy. DNA-PAINT offers excellent lateral resolution and versatile multiplexing, but its axial localization precision is typically 3-5 times poorer, limiting quantitative 3D imaging. Here, we present MIET-PAINT, which combines DNA-PAINT with Metal-Induced Energy Transfer (MIET) to overcome this limitation. We implement MIET-PAINT on both wide-field fluorescence lifetime and confocal TCSPC platforms. Wide-field MIET-PAINT enables robust, multiplexed imaging of focal adhesion proteins and actin in fixed cells. To address the lateral resolution limits of lifetime cameras, we further developed confocal MIET-PAINT, which leverages optical background rejection and high-efficiency SPAD detection. This modality achieves [~]12 nm lateral precision and resolves the 3D architecture of microtubules, vimentin, and actin with high fidelity. MIET-PAINT thus unites nanometer-scale axial accuracy with the multiplexing versatility of DNA-PAINT, establishing a powerful tool for quantitative 3D cell biology.

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↗

bioRxiv2023

Single-protein optical holography

Light scattering by nanoscale objects is a fundamental physical property defined by their scattering cross-section and thus polarisability. Over the past decade, a number of studies have demonstrated single molecule sensitivity, by imaging the interference between coherent scattering from the object of interest with a reference field. This approach has enabled mass measurements of single biomolecules in solution owing to the linear scaling of the image contrast with the molecular polarisability. Nevertheless, all implementations to date based on a common-path interferometer cannot separate and independently tune the reference and scattered light field, prohibiting access to the rich toolbox available to holographic imaging. Here, we demonstrate comparable sensitivity using a non-common path geometry based on a dark-field scattering microscope, similar to a Mach-Zehnder interferometer. We separate the scattering and reference light into four parallel, inherently phase stable detection channels, delivering a five orders of magnitude boost in sensitivity in terms of scattering cross-section over the state-of-the-art, demonstrating detection and mass measurement of single proteins below 100 kDa. Amplitude and phase measurement yields direct information on sample identity and the first experimental determination of the polarisability of single biomolecules.

biophysics↗

bioRxiv2021

Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer

Over the last two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution. Similar to conventional optical microscopy, the axial resolution is by a factor three to five worse than the lateral resolution. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging which achieves an axial resolution down to nanometers. It exploits the distance dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of sub-cellular structures. Moreover, we employed spectral demixing for implementing dualcolor MIET-dSTORM that allows us to image and co-localize, in three dimensions, two different cellular structures simultaneously.

biophysics↗

bioRxiv2020

Confocal Laser-Scanning Fluorescence-Lifetime Single-Molecule Localisation Microscopy

Fluorescence lifetime imaging microscopy (FLIM) is an important technique that adds another dimension to the intensity and colour information of conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is STimulated Emission Depletion (STED) microscopy. In contrast, all Single-Molecule Localisation Microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine Fluorescence-Lifetime Confocal Laser-Scanning Microscopy (FL-CLSM) with SMLM for realising single-molecule localisation-based fluorescence-lifetime super-resolution imaging (FL-SMLM). Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localised molecules. We validate our technique by applying it to direct STochastic Optical Reconstruction Microscopy (dSTORM) and Points Accumulation for Imaging in Nanoscale Topography (PAINT) imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

biophysics↗