Spatiotemporal Heterogeneity of κ-Carrageenan Gels Investigated via Single-Particle-Tracking Fluorescence Microscopy

Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions have remained elusive. Here, we use single-particle-tracking fluorescence microscopy (sptFM) to quantitatively describe κ-carrageenan gels. Infusing fluorescent probes into fully gelated κ-carrageenan hydrogels resulted in two distinct diffusional behaviors. Obstructed self-diffusion of the probes revealed that the coarse network consists of κ-carrageenan strands with a typical diameter of 3.2 ± 0.3 nm leading to a nanoprobe diffusion coefficient of ∼1-5 × 10-12 m2/s. In the dense network regions, we found a fraction with a largely decreased diffusion coefficient of ∼1 × 10-13 m2/s. We also observed dynamic exchange between these states. The computation of spatial mobility maps from the diffusional data indicated that the dense network regions have a characteristic diameter of ∼1 μm and show mobility on the second-to-minute timescale. sptFM provides an unprecedented view of spatiotemporal heterogeneity of hydrogel networks, which we believe bears general relevance for understanding transport and release of both low- and high-molecular weight solutes.</p

Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales

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Forster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multiscale molecular rulers

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Forster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble

Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

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

Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.</p

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Single-molecule localization microscopy as an emerging tool to probe multiscale food structures

Optical microscopy is an indispensable tool to characterize the microstructure of foods at ambient conditions. Depending on both the wavelength of light used to illuminate the sample and the opening angle of the microscope objective, the achievable resolution is limited to around 200 nm. This so-called classical diffraction limit implies that smaller structural features cannot be resolved or separated from each other. As many food structures are ultimately defined by the molecular interactions of single proteins or single molecules, the classical resolution is insufficient to reveal structural details in the (tens of) nanometer range. Intriguingly, recent advancements in imaging techniques originating mostly in the (biomedical) life sciences have been closing the gap, pushing the resolution towards true molecular resolution. In this perspective, we want to highlight some of these emerging techniques and provide an outlook on potential future applications

Open hardware in microscopy

The field of microscopy has been empowering humankind for many centuries by enabling the observation of objects that are otherwise too small to detect for the naked human eye. Microscopy techniques can be loosely divided into three main branches, namely photon-based optical microscopy, electron microscopy, and scanning probe microscopy with optical microscopy being the most prominent one. On the high-end level, optical microscopy nowadays enables nanometer resolution covering many scientific disciplines ranging from material sciences over the natural sciences and life sciences to the food sciences. On the lower-end level, simplified hardware and openly available description and blueprints have helped to make powerful microscopes widely available to interested scientists and researchers. For this special issue, we invited contributions from the community to share their latest ideas, designs, and research results on open-source hardware in microscopy. With this collection of articles, we hope to inspire the community to further increase the accessibility, interoperability, and reproducibility of microscopy. We further touch on the standardization of methodologies and devices including the use of computerized control of data acquisition and data analysis to achieve high quality and efficiency in research and development