Biology subjects

Yserentant, K.

Publications and source records attributed to Yserentant, K..

4 recordsLinked to original sources

bioRxiv2020

Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffractional resolution.

biophysics↗

bioRxiv2020

Photobleaching step analysis for robust determination of protein complex stoichiometries

The composition of cellular structures on the nanoscale is a key determinant of macroscopic functions in cell biology and beyond. Different fluorescence single-molecule techniques have proven ideally suited for measuring protein copy numbers of cellular structures in intact biological samples. Of these, photobleaching step analysis poses minimal demands on the microscope and its counting range has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost. Here, we present a comprehensive framework for photobleaching step analysis, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness and photostability. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we benchmark the framework with experimental data acquired on DNA origami labeled with defined fluorophore numbers to demonstrate counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis for robust protein counting by counting labelled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

biophysics↗

bioRxiv2020

Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labelling via bioorthogonal tetrazine chemistry confers minimal label size and rapid labelling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and fluorophore. Two synthetic routes give access to a series of cell permeable and impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as dominant excited state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multi-colour and super-resolution STED and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

cell biology↗

bioRxiv2020

A chemical strategy to control protein networks in vivo

Direct control of protein interaction by chemically induced protein proximity (CIPP) holds great potential for cell- and synthetic biology as well as therapeutic applications. However, toxicity, low cell-permeability and lack of orthogonality currently limit the use of available chemical inducers of proximity (CIP). We present Mandi, a novel CIP and demonstrate its applicability in cell culture systems as well as living organisms for protein translocation, protein network shuttling and manipulation of endogenous proteins.

synthetic biology↗