Biology subjects

Meub, M.

Publications and source records attributed to Meub, M..

3 recordsLinked to original sources

bioRxiv2023

Fast, whole-cell DNA-PAINT imaging with dual-labeled self-quenched imager probes

Elucidating the interaction between membrane proteins and antibodies requires fast whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-PAINT achieves molecular resolution but is practically restricted to two-dimensional imaging due to long acquisition times. Here, we introduce two-dye imager (TDI) probes, manifesting negligible background and amplified fluorescence signal upon transient binding, enabling [~]15-fold faster imaging. Using a combination of TDI-DNA-PAINT and LLS microscopy on B cells, we reveal the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibody rituximab (RTX), unperturbed by surface effects. Our results demonstrate that B cells become polarized, and microvilli stabilized by RTX binding. These findings, we believe, will aid rational design of improved immunotherapies targeting tumor-associated antigens.

biophysics↗

bioRxiv2022

Sub-10 nm fluorescence imaging

Advances in superresolution microscopy demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10 nm spatial resolution in biological samples remains challenging. Here, we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10 nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range. In combination with genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores photoswitching fingerprint analysis enables sub-10 nm resolution fluorescence imaging in cells.

biophysics↗

bioRxiv2021

Mapping densely distributed membrane receptors in blood platelets with expansion microscopy

Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed GPIIb/IIIa receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of GPIIb/IIIa clusters in resting platelets, which are not affected by platelet activation indicating that they contribute to the rapid platelet response during platelet clotting. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/431449v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@c4e38forg.highwire.dtl.DTLVardef@37c1adorg.highwire.dtl.DTLVardef@8c8597org.highwire.dtl.DTLVardef@196bd69_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗