Conference Agenda

In the crowded plasma membrane of a living cell, for instance, integral membrane proteins (IMP) can organize into multi-IMP assemblies known as transport metabolons. In these complexes, an enzyme is physically (transiently) coupled to membrane transporters, accelerating metabolite flux by bypassing free diffusion limitations. The bicarbonate metabolon, in which carbonic anhydrase IX (CAIX) plays a crucial role, is essential for pH regulation.

Here we will present our research results that leverages advanced SM localization microscopy (SMLM) with SM tracking to unravel the dynamic behavior of CAIX in live cell membranes using these CAIX-targeting probes :

(1) Small-molecule ligands with tunable affinity and sulforhodamine B labeling ,

(2) Fab antibody fragments labeled with ATTO647N/abberiorCAGE635 targeting the CAIX PG domain,

(3) Nanodiamonds with nitrogen-vacancy centers functionalized with CAIX ligands.

Live-cell SMT experiments were performed in wild-type and CAIX knock-out HeLa cells. We quantified CAIX localization densities, diffusion coefficients, and track lengths under normoxic, hypoxic and over-expression conditions, revealing cell-to-cell heterogeneity in CAIX expression and mobility. Knock-out controls confirmed probe specificity. Our ongoing work extends this approach to primary cancer cells from patients, aiming to correlate CAIX dynamics with clinical phenotypes. This study provides new insights into the molecular mechanisms of CAIX in cancer.

Super-resolution fluorescence microscopy has emerged as a powerful tool for investigating biological structures at the nanoscale, well beyond the diffraction limit of classical optical imaging. Among super-resolution techniques, single-molecule localization microscopy achieves this by sequentially imaging and localizing individual fluorescent emitters, with methods such as DNA-PAINT reaching resolutions below 10 nm through transient binding of fluorescently labeled oligonucleotides. Such experiments place stringent demands on instrumentation: long acquisition times require active sample drift correction across all axes, multi-color imaging benefits from simultaneous dual-channel detection, and flexible software pipelines are essential for real-time feedback and custom workflows.

We present the latest developments in the miEye and microEye open-source ecosystem, a modular benchtop single-molecule localization microscopy platform that combines cost-effective hardware with a comprehensive Python-based software stack. Hardware updates include improved mechanical modularity, three-axis active drift correction via a dedicated near-infrared detection path, and dual visible-channel emission detection. The microEye software has been restructured around a unified hardware abstraction layer with Pycro-Manager integration and a modular architecture designed for future real-time analysis workflows. System capabilities are demonstrated through imaging DNA origami nano-ruler samples.

A dual-branch method for limited-view PAM combines PAFI-based temporal statistics with SMLM-inspired localization of dynamic RBC signals. It reduces structural loss, boundary blur, and discontinuity, while improving vessel continuity and fine-detail visualization in curved and view-limited regions.

Bacteria are constantly facing various outside threats, such as bacteriophages and parasitic plasmids. Due to the rapid advance of bioinformatic tools, more than hundred different bacterial defense system candidates have been proposed to date. The canonical bacterial defense systems – restriction-modification, CRISPR-Cas and prokaryotic Argonaute protein-based – were extensively studied using bulk biochemical assays and diverse structural techniques. However, single-molecule characterization of these systems’ effectors is still lacking, which could aid in elucidating their complete molecular mechanisms.

Here, we investigated the DNA-interaction dynamics of three fundamentally different bacterial defense system effector proteins at the single-molecule level. We used single-molecule FRET to detect and monitor in cis and in trans interactions between DNA and BfiI restriction endonuclease from Bacillus firmus. Next, by combining single-molecule fluorescence microscopy with DNA flow-stretch assay, we revealed filamentation-based mechanism of action of the novel anti-plasmid defense system SPARDA from Xanthobacter autotrophicus strain Py2. Finally, we performed dSTORM-based single-molecule tracking of Streptococcus pyogenes Cas9 diffusion to probe its target search in living Escherichia coli cells. Our findings should expand the mechanistic understanding about these key effector proteins and pave the way for effectively applying them as optimized or novel molecular tools in genome engineering, biotechnology and biomedicine.

Super-Resolution Microscopy is a key technology across many disciplines, enabling the observation of 3D structural details with nanometer precision. Further breakthroughs have extended the capabilities, resolving 3D orientation and spectral signatures of dipole emitters. However, those capabilities require complex optical systems demanding exceptional stability and specialized expertise in optics. To overcome this, we propose a simple bi-focal system based on birefringence and exploiting polarization as well as dispersion effects, to simultaneously determine 3D position, orientation and spectral signature of nanoprobes. Notably, extracting this set of measurands requires no modifications to a standard wide-field microscope other than the use of a birefringent microscope slide.