Published: Adapting cryogenic correlative light and electron microscopy (cryo-CLEM) for food oxidation studies

S.Yang, M. Takeuchi, R. Joosten, J. van Duynhoven, H. Friedrich, J. Hohlbein, Food Structure, 40, 100365, 2024, [link], preprint on chemRxiv, [link]

Lipid oxidation is a major cause of product deterioration in protein stabilised oil-in-water food emulsions. The impact of protein emulsifiers on lipid oxidation and the stability depends on the specific type of protein emulsifiers used and the redox conditions in the emulsion. However, the exact impact of these protein emulsifiers at the oil-water interface on lipid oxidation and the mechanism of lipid-protein co-oxidation are currently unknown. Here, we developed a cryo-correlative light and electron microscopy (cryo-CLEM) platform for co-localising the oxidation of lipids and proteins. For this first implementation of cryo-CLEM for food oxidation studies we optimised specifically the part of cryo-fluorescence microscopy (cryo-FM) by adding parts that prevent fogging on the sample and enable homogeneous laser illumination. We showed that lipid oxidation in food emulsions can be observed at cryogenic temperature using fluorescence imaging of the fluorophore BODIPY 665/676 that we employed earlier as a lipid oxidation sensor at room temperature. Using cryo-transmission electron microscopy (cryo-TEM), we observed that more protein aggregates are found at the droplet interfaces in oxidized emulsions compared to fresh emulsions. Our cryo-CLEM platform paves the way for future cryo-correlative oxidation studies of food emulsions.

Published: Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking

K.J.A. Martens, B. Turkowyd, J. Hohlbein, U. Endesfelder, Nature Methods, 2024, [link], preprint on bioRxiv, [link]

Viscosity is a fundamental property of liquids. It determines transport and diffusion of particles in solution. Nonetheless, it is an open question how a gradient of viscosity – causing a gradient in diffusivity – can lead to viscophoretic transport, i.e., directed transport of particles and molecules in solution. Here, we determine viscophoretic drift experimentally. We generate steep, stable viscosity gradients in a microfluidic device and image transport of suspended nanoparticles in these gradients using high-resolution microscopy. We observe high viscophoretic drift velocities which significantly exceed theoretical predictions. In addition, we demonstrate a new method for trapping and concentrating particles by using the interplay of viscophoresis and diffusiophoresis. We believe that a quantification of viscophoresis will advance the understanding and application of transport processes of gradients of viscosity occurring in very diverse fields such as cell biology, chromatography, and membrane technology.

Pre-print: Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a

L. Olivi, C. Bagchus, V. Pool, E. Bekkering, K. Speckner, W. Wu, K.J.A Martens, J. van der Oost, R. Staals, J. Hohlbein, pre-print on bioRxiv, [link]

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search

Published: Open hardware in microscopy

Open hardware in microscopy, J. Hohlbein and S. Faez, HardwareX, 15, e00473, 2023, [link]

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.

Published: Response to “Liposome vesicle cannot be formed in non-aqueous phase”

S. Yang, M. Takeuchi, H. Friedrich, J.P.M. van Duynhoven, and J. Hohlbein, Food Chemistry, 439, 138087, 2024, [link]

In a recent letter to the editor Prof Khosravi-Darani responded to our paper ‘’Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales’’. In our work, we observed liposomes in the continuous phase of mayonnaise. In the letter the objection was made that liposomes cannot be formed in a non-aqueous phase which, however, was not argued in our publication. As mayonnaise is an oil-in-water (O/W) emulsion and its continuous phase is aqueous, liposomes may be observed in this phase. Therefore, the objection from Prof Khosravi-Darani does not apply to our work.

Pre-print: Droplet size dependency and spatial heterogeneity of lipid oxidation in whey protein isolate-stabilized emulsions

S. Yang, S. ten Klooster, K. Nguyen, M. Hennebelle, C. Berton-Carabin, K. Schroën, J. van Duynhoven, J. Hohlbein, pre-print on chemRxiv, [link]

Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical formation. Next, we assessed the impact of microfluidic and colloid mill emulsification on local protein and lipid oxidation kinetics in whey protein isolate (WPI)-stabilized emulsions. For that purpose, we also used BODIPY 581/591 C11 and CAMPO-AFDye 647 as colocalisation markers for lipid and protein oxidation. The polydisperse emulsions showed an inverse relation between droplet size and lipid oxidation rate. Further, we observed less protein and lipid oxidation occurring in similar sized droplets in monodisperse emulsions. This observation was linked to more heterogeneous protein packing at the droplet surface during colloid mill emulsification, resulting in larger inter-droplet heterogeneity in both protein and lipid oxidation. Our findings indicate the critical roles of emulsification methods and droplet sizes in understanding and managing lipid oxidation.

Pre-print: Viscophoretic particle transport

V. Khandan, V. Boerkamp, A. Jabermoradi, M. Fontana, J. Hohlbein, E. Verpoorte, R.C. Chiechi, K. Mathwig, 2022, pre-print on arXiv, [link]

Viscosity is a fundamental property of liquids. It determines transport and diffusion of particles in solution. Nonetheless, it is an open question how a gradient of viscosity – causing a gradient in diffusivity – can lead to viscophoretic transport, i.e., directed transport of particles and molecules in solution. Here, we determine viscophoretic drift experimentally. We generate steep, stable viscosity gradients in a microfluidic device and image transport of suspended nanoparticles in these gradients using high-resolution microscopy. We observe high viscophoretic drift velocities which significantly exceed theoretical predictions. In addition, we demonstrate a new method for trapping and concentrating particles by using the interplay of viscophoresis and diffusiophoresis. We believe that a quantification of viscophoresis will advance the understanding and application of transport processes of gradients of viscosity occurring in very diverse fields such as cell biology, chromatography, and membrane technology.

Published: Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

M. Fontana, M. Roosjen, I. Crespo García, W. van den Berg, M. Malfois, R. Boer, D. Weijers, and J. Hohlbein, PNAS, 120, e2219916120, 2023, [link], pre-print on bioRxiv, [link]

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF–ARF and ARF–DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Published: Enabling spectrally resolved single-molecule localization microscopy at high emitter densities

K.J.A. Martens, M. Gobes, E. Archontakis, N. Zijlstra, L. Albertazzi, and J. Hohlbein, Nano Letters, 22, 8618, 2022, [link], pre-print on bioRxiv, [link]

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1st diffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

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

S. Yang, M. Takeuchi, H. Friedrich, J.P.M. van Duynhoven, and J. Hohlbein, Food Chemistry, 402, 134417, 2023, [link], preprint on chemRxiv, 2022, [link]

In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles acting as a main emulsifier. Here, we monitored oxidation and the concomitant aggregation of LDLs by bright-field light microscopy and cryogenic transmission electron microscopy. We further probed the formation of protein radicals and protein oxidation by imaging the accumulation of a water-soluble fluorescent spin trap and protein autofluorescence. The effect of variation of pH and addition of EDTA on the accumulation of the spin trap validated that protein radicals were induced by lipid radicals. Our data suggests two main pathways of oxidative protein radical formation in LDL particles: (1) at the droplet interface, induced by lipid free radicals formed in oil droplets, and (2) in the continuous phase induced by an independent LDL-specific mechanism.