Biology subjects

Jungblut, M.

Publications and source records attributed to Jungblut, M..

6 recordsLinked to original sources

bioRxiv2026

Programmable protein reference standards for benchmarking sub-10-nm fluorescence microscopy

Fluorescence microscopy is increasingly used to measure molecular organization at length scales where labeling, photophysics and sample preparation can dominate quantitative accuracy. Reference standards are therefore needed that combine defined nanoscale geometry with a protein-like environment and compatibility with biological imaging. Here we introduce circular tandem repeat protein as programmable protein standards for benchmarking sub-10-nm fluorescence microscopy. Using genetic code expansion and bioorthogonal labeling, we generated compact protein rings carrying up to six labeling sites. Photoswitching fingerprint analysis revealed geometry-dependent localization accumulation and blinking kinetics, demonstrating that short-range fluorophore interactions can be assessed as measurable benchmark parameters. DNA-PAINT confirmed accessible docking sites and programmed valency at the single-particle level. We further established recombinant tethering and genetically encoded membrane display, extending the standards to cellular environments. cTRP PicoRulers were also compatible with expansion microscopy. Together, cTRPs provide a modular protein-based platform for evaluating molecular-scale imaging performance in purified and cellular environments.

biophysics↗

bioRxiv2026

Nanoscale imaging of expanded cells and proteins with spontaneously blinking dyes

Expansion microscopy (ExM) enables nanoscale imaging on standard microscopes, but combining ExM with single-molecule localization microscopy (SMLM) remains difficult, owing to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy method that allows SMLM with spontaneously blinking dyes in 6-14x expanded samples, without re-embedding. We demonstrate nanometer-resolution imaging by resolving the organization of the nuclear pore complex (NPC) and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen (PCNA).

biophysics↗

bioRxiv2025

Integrated 3D Light-Sheet and 2D Multiplex Imaging for Deep Histological Profiling of a Somatic Mouse Glioblastoma Model

Glioblastoma is a devastating brain cancer. Despite intense research, patient survival has not significantly increased over the past decades and efficient treatment is currently not available. Therefore, the fundamental understanding of the disease, based on the development of relevant animal models, combined with the development of efficient tools for their deep analysis, represents a priority. Neural Stem cells in the subventricular zone of the forebrain have been identified as cells of origin for glioblastoma, leading to the development of new somatic lineage models based on in vivo brain electroporation. While such models have been characterized in depths by sequencing approaches, systematic histological analyses are currently scarce. Here we present the multimodal histological characterization of a transgenesis independent somatic glioblastoma model in mice. Using 3D light sheet imaging we demonstrate that the model is highly reproducible, allowing quantitative evaluation of tumor growth over large cohorts. Using multiplex imaging by MICS technology we systematically characterize the cellular landscape and molecular composition of the induced tumors, as well as their micro- and macro-environments, and provide a resource of mouse compatible antibodies for cancer research. Finally, we use the model to show that tissue clearing and 3D light sheet microscopy of whole brains can be combined with subsequent multiplex imaging, allowing deep spatial characterization of the tumor proteome in pre-identified brain regions.

cancer biology↗

bioRxiv2025

Resolving protein organization in cells with nanometer resolution

Advances in super-resolution microscopy have outpaced molecular labeling methods, impeding nanometer-scale imaging of endogenous multiprotein complexes in cells. To overcome these limitations, we developed an expansion microscopy (ExM) approach based on double-homogenized hydrogels that enables direct stochastic optical reconstruction microscopy (dSTORM) of 7-8-fold expanded immunolabeled samples. The resulting [~]4-fold increase in effective immunolabeling density resolved the 8 nm distance between neighboring -tubulin molecules in microtubules and the polyhedral lattice in clathrin-coated pits with nanometer precision in cells. Two-color Ex-dSTORM further revealed the molecular organization of RIM scaffolding protein and Munc13-1, an essential synaptic vesicle priming protein, in ring-like structures with diameters of 40-45 nm at the presynapse in hippocampal neurons. Our results demonstrate that Ex-dSTORM resolves the molecular organization of endogenous multiprotein complexes with nanometer spatial resolution in genetically unmodified cells. Thus, it provides a versatile method for the investigation of molecular protein distributions in their physiologically relevant context. One-Sentence SummaryThe optimized combination of dSTORM and double TREx ExM resolves previously unresolvable protein assemblies in cells

biophysics↗

bioRxiv2023

Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early, late and recycling endosomes. To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for early, late, and recycling endosomes. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

cell biology↗

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↗