Punter, M. | Search bioRxiv

bioRxiv2023

Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to infer the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells. We find that this observation is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging.

biophysics↗

bioRxiv2018

Self-healing dyes for super-resolution microscopy

In recent years optical microscopy techniques have emerged that allow optical imaging at unprecented resolution beyond the diffraction limit. Up to date, photostabilizing buffers are the method of choice to realize either photoswitching and/or to enhance the signal brightness and stability of the employed fluorescent probes. This strategy has, however, restricted applicability and is not suitable for live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores with intramolecular photostabilization in super-resolution microscopy with targeted (STED) and stochastic readout (STORM). The overall goal of the study was to improve the spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. Due to its past superior performance we identified ATTO647N-photostabilizer conjugates as suitable candidates for STED microscopy. We characterize the photostability and resulting performance of NPA-ATTO647N oligonucleotide conjugates in STED microscopy. We find that the superior photophysical performance results in optimal STED imaging and demonstrate the possibility to obtain single-molecule fluorescent transients of individual fluorophores while illuminating with both the excitation- and STED-laser. Secondly, we show an analysis of photoswitching kinetics of self-healing Cy5 dyes (comprising TX, COT and NPA stabilizers) in the presence of TCEP- and cysteamine, which are typically used in STORM microscopy. In line with previous work, we find that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.

biophysics↗