, 2017, [
Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels. A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.
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We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.
For the software implementation into thunderSTORM, please follow the [link].
…who joins the group for his MSc thesis. In collaboration with Dr. Klaus Mathwig (Pharmacy, Groningen), Dr. Peter van Baarlen (Host-Microbe Interactions, Wageningen) and Simon van der Els (NIZO food research and HMI, Wageningen), he will work towards monitoring bacterial conjugation in real time and at the single-cell level.
K. Mathwig, C. Fijen, M. Fontana, S. G. Lemay and J. Hohlbein, Procedia Technology, 27, 141, 2017, [link]
We introduce a nanofluidicmixing device
entirely fabricated in glass for the fluorescence detection of single molecules. The design consists of a nanochannel T-junction and allows the continuous monitoring of chemical or enzymatic reactions of analytes as they arrive from two independent inlets. The fluorescently labeled molecules are tracked before, during and after they enter the mixing region, and their reactions with each other are observed by means of optical readout such as Förster Resonance Energy Transfer (FRET). Our method can be used for analyzing the kinetics of DNA annealing in a high-parallelized fashion.
Good news! The proposal “Lipid Oxidation Control in Food Emulsions Enabled by Natural Strategies” submitted by Harry Gruppen (WUR) has been awarded a CHIPP grant by The Innovation Fund for Chemistry [link].
Our lab will head the work package titled “Localising oxidation: interfaces and transport” and a PhD position will be available in 2018.
Here the specs: 50k frames, 850k localisations, 6 min for data analysis. Many thanks to Sander Baas, Koen Martens and Arjen Bader, with cells provided by Gert-Jan Bakker and Ben Joosten (both Radboudumc, Nijmegen).
Good news! Our proposal “Towards visualizing gene transfer at the single cell level using microfluidic devices” has been granted by the Innovation Program Microbiology, an initiative from the Laboratory of Microbiology at Wageningen University and Research.
In collaboration with Dr. Klaus Mathwig (Pharmacy, Groningen), Dr. Peter van Baarlen (Host-Microbe Interactions, Wageningen) and Simon van der Els (NIZO food research and HMI, Wageningen), we aim to monitor bacterial conjugation, in which DNA is transferred from a donor cell to a recipient cell either via conjugative plasmids or via integrative conjugative elements, in real time and at the single-cell level.