In situ generation and atomic scale imaging of slip traces with atomic force microscopy

We have designed, constructed, and tested a three-point bending system for in situ studies of slip in ionic crystals with an atomic force microscope (AFM). The work is aimed at developing a novel instrumental attachment for an in situ study of plastic deformation. The bending system is installed inside the optical head of the AFM on top of the piezoelectric scanner. Since the bending should not obstruct scanning, a piezocrystal is used for bending as well as an external stepper motor, which is connected with a screw in the bending system via a flexible shaft. The bending system performs over a relatively wide, continuous deflection range. The operation of the three-point bending system is illustrated by experiments on an ionic material in which the effect of macroscopic bending is demonstrated at an atomic scale.

Quantitative analysis of surface wave patterns of Min proteins

The Min protein system is arguably the best-studied model system for biological pattern formation. It exhibits pole-to-pole oscillations in E. coli bacteria as well as a variety of surface wave patterns in in vitro reconstitutions. Such Min surface wave patterns pose particular challenges to quantification as they are typically only semi-periodic and non-stationary. Here, we present a methodology for quantitatively analysing such Min patterns, aiming for reproducibility, user-independence, and easy usage. After introducing pattern-feature definitions and image-processing concepts, we present an analysis pipeline where we use autocorrelation analysis to extract global parameters such as the average spatial wavelength and oscillation period. Subsequently, we describe a method that uses flow-field analysis to extract local properties such as the wave propagation velocity. We provide descriptions on how to practically implement these quantification tools and provide Python code that can directly be used to perform analysis of Min patterns.BN/Cees Dekker La

Direct observation of independently moving replisomes in Escherichia coli

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in widened E. coli cells. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.</p

AutoStepfinder: A fast and automated step detection method for single-molecule analysis

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour.BN/Chirlmin Joo LabBN/Technici en AnalistenBN/Cees Dekker La