Biology subjects

Jans, D. C.

Publications and source records attributed to Jans, D. C..

4 recordsLinked to original sources

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

Super-resolution microscopy of mitochondrial mRNAs

Mitochondria contain their own DNA (mtDNA) and a dedicated gene expression machinery. As the mitochondrial dimensions are close to the diffraction limit of classical light microscopy, the spatial distribution of mitochondrial proteins and in particular of mitochondrial mRNAs remains underexplored. Here, we establish single-molecule fluorescence in situ hybridization (smFISH) combined with STED and MINFLUX super-resolution microscopy (nanoscopy) to visualize individual mitochondrial mRNA molecules and associated proteins. STED nanoscopy revealed the spatial relationships between distinct mRNA species and proteins such as the RNA granule marker GRSF1, demonstrating adaptive changes in mRNA distribution and quantity in challenged mammalian cells and patient-derived cell lines. Notably, STED-smFISH showed the release of mRNAs during apoptosis, while MINFLUX revealed the folding of the mRNAs into variable shapes, as well as their spatial proximity to mitochondrial ribosomes. These protocols are transferable to various cell types and open new avenues for understanding mitochondrial gene regulation in health and disease.

cell biology↗

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

Endogenous BAX and BAK form mosaic rings of variable size and composition on apoptotic mitochondria

One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same temporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wildtype cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK-single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings corroborates the toroidal apoptotic pore model.

cell biology↗