Biology subjects

Enderlein, J.

Publications and source records attributed to Enderlein, J..

At least 19 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↗

bioRxiv2026

Metal-induced energy transfer uncovers activation-induced axial reorganization of signaling complexes inside cells

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domain (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of [~]1 nm. Using the prototypic, biomedically relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results demonstrate the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

biophysics↗

bioRxiv2026

Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing

Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

cell biology↗

bioRxiv2025

Self-blinking dye restores efficient use of nanobodies in single-molecule localization microscopy.

Direct stochastic optical reconstruction microscopy (dSTORM) relies on controlled fluorophore blinking to achieve nanometer-scale resolution, yet the fields benchmark dye, Alexa Fluor 647, underperforms when conjugated to nanobodies, limiting the practical use of minimal-linkage labeling strategies. Here, we show that the self-blinking dye JF635b overcomes this limitation by maintaining robust, photostable blinking upon conjugation to nanobodies under buffer-independent conditions. This enables reliable single-molecule localization microscopy without the need for complex switching buffers. Using JF635b-labeled nanobodies, we demonstrate consistent performance across multiple imaging modalities, including wide-field dSTORM, fluorescence lifetime SMLM, and MINFLUX nanoscopy, achieving localization precisions from [~]15 nm down to the sub-nanometer regime. In addition, JF635b supports long-term sample preservation and efficient blinking even in pure water, enabling minimally perturbative imaging conditions. Together, these results establish self-blinking dSTORM as a robust and accessible platform for quantitative nanoscopy across experimental contexts.

biophysics↗

bioRxiv2025

A versatile microfluidics platform for enhanced multi-target super-resolution microscopy

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is the capacity for multiplexing, which is achieved through a process known as Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, and MINFLUX platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells and, importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications highlight the unique power of our platform to extend super-resolution multiplexing into physiologically relevant systems, thereby opening new avenues for biomedical research.

biophysics↗

bioRxiv2025

Plasmonic Nanocavity-Based Method for Measuring Electric Charges in Solution

Electric charges play a fundamental role in shaping the structure, function, and interactions of biomolecules, yet precisely measuring these charges at the single-molecule level remains a significant technical challenge. Here, we introduce a novel experimental methodology that utilizes plasmonic nanocavities to quantify molecular electric charges in solution with high sensitivity. Our approach exploits an externally applied electric field to induce the spatial redistribution of charged molecules confined within a planar metallic nanocavity, while simultaneously leveraging nanocavity-induced fluorescence lifetime modulation as a highly sensitive readout. We demonstrate the feasibility of this method through proof-of-concept experiments, where we measure the fluorescence lifetimes of positively and negatively charged fluorescent dye molecules as a function of the applied electric field across the cavity. The experimental results are validated through a rigorous theoretical framework, incorporating statistical thermodynamics and electrodynamic modeling to accurately describe the observed data. The proposed method offers a calibration-free, experimentally simple, and rapid alternative to existing charge measurement techniques, opening new avenues for precise quantification of molecular electric charges down to the single-molecule level.

biophysics↗

bioRxiv2024

Super-Resolution Axial Imaging for Quantifying Piconewton Traction Forces in Live-cells

Cell mechanics play a pivotal role in regulating numerous biological processes. While super-resolution microscopy enables the imaging of cellular forces in the lateral dimension with sub-10-nanometer resolution, achieving comparable resolution along the axial dimension remains a significant challenge. In this study, we combine metal-induced energy transfer (MIET) imaging with novel DNA-hairpin-based molecular tension probes (MIET-MTP) to map integrin-mediated mechanical forces with nanometer precision in the axial direction. MIET-MTP allows for the simultaneous observation of both the plasma membrane and forceexerting molecules in the axial dimension. Using this approach, we mapped axial integrin tension in focal adhesions and podosomes, alongside their corresponding plasma membrane height profiles, offering detailed insights into the structures involved in force transmission.

biophysics↗

bioRxiv2024

Measuring 3-dimensional dynamics of membrane in living cell with metal-induced energy transfer imaging/spectroscopy

The dynamic behavior of cellular membranes underpins essential biological processes, including signal transduction, intracellular trafficking, and mechanotransduction. However, simultaneously quantifying lateral molecular diffusion and vertical membrane fluctuations in live cells remains challenging. Here, we present dynamic metal-induced energy transfer spectroscopy (dynaMIET), which integrates metal-induced energy transfer with fluorescence correlation spectroscopy to resolve three-dimensional membrane dynamics with nanometer axial sensitivity and microsecond temporal resolution. dynaMIET enables concurrent measurement of lateral diffusion and vertical undulations within a single acquisition. We validate the method using simulations and model membranes and demonstrate its robustness in living cells, applying it to the plasma membrane, endoplasmic reticulum, and nuclear envelope. By capturing both molecular mobility and membrane fluctuations, dynaMIET provides a powerful, non-invasive tool for probing membrane mechanics and organization. This advance opens new avenues for studying membrane-associated phenomena in health and disease, including cancer cell mechanics, protein-membrane interactions, and organelle dynamics.

biophysics↗

bioRxiv2024

Scattering-Based Super-Resolution Optical Fluctuation Imaging

Super-resolution optical imaging has become a prominent tool in life and material sciences, allowing one to decipher structures at increasingly greater spatial detail. Among the utilized techniques in this field, super-resolution optical fluctuation imaging (SOFI) has proved to be a valuable approach. A major advantage of SOFI is its less restrictive requirements for generating super-resolved images of neighboring nanostructures or molecules, as it only assumes that the detected fluctuating light from neighboring emitters is statistically uncorrelated, but not necessarily separated in time. While most optical super-resolution microscopies depend on signals obtained from fluorescence, they are limited by photobleaching and phototoxicity. An alternative source for optical signals can be acquired by detecting the light scattered from molecules or nanoparticles. However, the application of coherent scattering-based imaging modalities for super-resolution imaging has been considerably limited compared to fluorescence-based modalities. Here, we develop scattering-based super-resolution optical fluctuation imaging (sSOFI), where we utilize the rotation of anisotropic particles as a source of fluctuating optical signals. We discuss the differences in the application of SOFI algorithms for coherent and incoherent imaging modalities, and utilize interference microscopy to demonstrate super-resolution imaging of rotating nanoparticle dimers. We present a theoretical analysis of the relevant model systems, and discuss the possible effects of cusp artifacts and electrodynamic coupling between nearby nano-scatterers. Finally, we apply sSOFI as a label-free novelty filter that highlights regions with higher activity of biomolecules and demonstrate its use by imaging membrane protrusions of live cells. Overall, the development of optical super-resolution approaches for coherent scattering-based imaging modalities, as described here, could potentially allow for the investigation of biological processes at temporal resolutions and acquisition durations previously inaccessible in fluorescence-based imaging.

biophysics↗

bioRxiv2024

Conformational dynamics of electrostatically charged Intrinsically Disordered Proteins

We experimentally and computationally study the dynamics of homogeneously electrically charged intrinsically disordered proteins (IDPs) in ionic solutions (aqueous solutions containing salt). Computationally, this is achieved by extending a recently introduced discrete elastic rod (DER) model to include electrostatic interactions. The respective model includes only three free parameters: bending stiffness, bond length, and hydrodynamic radius. These parameters were obtained by fitting the measured conformational dynamics of uncharged polypeptides as reported in a previous publication [8]. We compare our computational results with photo-electron transfer fluorescence correlation spectroscopy (PET-FCS) measurements of the conformational dynamics of the highly charged (-44 e-) intrinsically disordered protein Prothymosin . We here report on an agreement between the loop-closing rates obtained from our computational model and the respective experimental measurements, with rate values significantly slower than those observed for uncharged IDPs.

biophysics↗

bioRxiv2024

Variational calculus approach to Zernike polynomials with application to fluorescence correlation spectroscopy

Zernike polynomials are a sequence of orthogonal polynomials that play a crucial role in optics and in particular in modeling microscopy systems. Introduced by Frits Zernike in 1934, they are particularly useful in expressing wavefront aberrations and thus imperfections of imaging systems. However, their origin and properties are rarely discussed and proven. Here, we present a novel approach to Zernike polynomials using variational calculus, and apply them to describe aberrations in fluorescence microscopy. In particular, we model the impact of various optical aberrations on the performance of one-photon and two-photon excitation fluorescence microscopy. SIGNIFICANCEThis manuscript explores the mathematical derivation of Zernike polynomials and highlights their critical role in describing optical wavefronts and aberrations, particularly in the domain of optical microscopy. Special emphasis is placed on their utility in simulating the effects of aberrations on one-photon and two-photon excitation fluorescence correlation spectroscopy.

biophysics↗

bioRxiv2024

Super-resolution going viral: T4 virus particles as perfect nature-designed 3D-Bio-NanoRulers

In the burgeoning field of super-resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the three-dimensional domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of three-dimensional subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities. Choosing the optimal standard poses a considerable challenge, necessitating, among other attributes, precisely defined geometry and the capability for specific labeling at sub-diffraction-limit distances. In this context, we introduce the use of the non-human-infecting virus, bacteriophage T4, as an effective and straightforward bio-ruler for three-dimensional super-resolution imaging. Employing DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) along with the technique of astigmatic imaging, we uncover the icosahedral capsid of the bacteriophage T4, measuring 120 nm in length and 86 nm in width, and its hollow viral tail. This level of detail in light microscopy represents a significant advancement. We further outline a simple protocol for the production and preparation of samples. Moreover, we explore the extensive potential of bacteriophage T4 as a multi-faceted 3D bio-ruler, proposing its application as a novel benchmark for three-dimensional super-resolution imaging in biological studies.

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↗

bioRxiv2024

Nanoscale architecture and dynamics of CaV1.3 channel clusters in cardiac myocytes revealed by single channel nanoscopy

The clustering of L-type calcium channels for functional regulation of intracellular calcium signaling remains poorly understood. Here we applied super-resolution imaging to study CaV1.3 channel clusters in human iPSC-derived atrial cardiomyocytes (hiPSC-aCM) to analyze subcellular localization, dimensions, architecture, and dynamics, which were largely unexplored previously. STimulated Emission Depletion (STED) imaging characterized the localization and structure of CaV1.3 channel clusters in living cardiomyocytes. DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) achieved true molecular resolution, revealing an irregular channel distribution with substantial spacing. Single Particle Tracking (SPT) showed that channels co-diffuse in confined and stationary membrane nanodomains. The cytosolic C-terminal tail of CaV1.3 by itself was found sufficient for cluster formation. In conclusion, our LTCC clustering studies demonstrate that CaV1.3 channel clusters consist of mobile individual channels inside defined membrane nanodomains, in contrast to previous models of dense channel packing.

cell biology↗

bioRxiv2023

Observation of E-cadherin Adherens Junctions Variation with Metal-induced Energy Transfer Imaging/Spectroscopy

Epithelial cadherin (E-cad) mediated cell-cell junctions play a crucial role in the establishment and maintenance of tissues and organs. In this study, we employed metal-induced energy transfer imaging and spectroscopy to investigate variations in intermembrane distance during adhesion between two model membranes adorned with E-cad. By correlating the measured intermembrane distances with the distinct E-cad junction states, as determined by their crystal structures, we probed the dynamic behavior and diversity of E-cad junctions across different binding pathways. Our observations led to the identification of a transient intermediate state referred to as the X-dimeric state and enabled a detailed analysis of its kinetics. We discovered that the formation of the X-dimer leads to significant membrane displacement, subsequently impacting the formation of other X-dimers. These direct experimental insights into the subtle dynamics of E-cad-modified membranes and the resultant changes in intermembrane distance provide novel perspectives on the assembly of E-cad junctions between cells. This knowledge en-hances our comprehension of tissue and organ development and may serve as a foundation for the development of innovative therapeutic strategies for diseases linked to cell-cell adhesion abnormalities. Significance StatementIn this study, we employed metal-induced energy transfer (MIET) imaging and spectroscopy to track variations in intermembrane distance during the adhesion of two membranes mediated by epithelial cadherin. Leveraging the high spatial resolution of MIET, we explored the dynamics of cadherins across various binding pathways. Furthermore, we successfully captured a transient intermediate state known as the X-dimer and revealed its ability to communicate with other X-dimers through membrane displacement. These discoveries offer valuable mechanistic insights into the dynamics of cadherin junctions.

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

Metal-induced energy transfer (MIET) imaging of cell surface engineering with multivalent DNA nanobrushes

The spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the inter-membrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of cell-cell interactions, such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with the MIET imaging/spectroscopy. Moreover, we successfully employ MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment does not only showcase the effectiveness of MIET as a powerful tool for accurately quantifying cell-cell interactions, but does also validate the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.

biophysics↗

bioRxiv2023

Measuring sub-nanometer fluctuations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy

Out-of-plane fluctuations, also known as stochastic displacements, of biological membranes play a crucial role in regulating many essential life processes within cells and organelles. Despite the availability of various methods for quantifying membrane dynamics, accurately quantifying complex membrane systems with rapid and tiny fluctuations, such as mitochondria, remains a challenge. In this work, we present a novel methodology that combines metal/graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to quantify out-of-plane fluctuations of membranes with simultaneous spatiotem-poral resolution of approximately one nanometer and one microsecond that is unprecedented. To validate the technique and spatiotemporal resolution, we measured bending undulations of model membranes. Furthermore, we demonstrate the versatility and applicability of MIET/GIET-FCS for studying diverse membrane systems, including the widely studied fluctuating membrane system of human red blood cells, as well as two unexplored membrane systems with tiny fluctuations, a pore-spanning membrane, and mitochondrial inner/outer membranes.

biophysics↗