Pre-print: 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, bioRxiv, 2022, [link]

Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing of local chemical environments by using solvachromatic fluorophores such as Nile Red. Until now, the widespread utilisation of sSMLM was hampered by several challenges: an increased complexity of the optical detection pathway, limited software solutions for data analysis, lower accessible emitter densities or smaller field-of-views, and overall compromised spatio-spectral resolution. Here, we present a low-cost implementation of sSMLM that addresses these challenges. Using a blazed, low-dispersion transmission grating positioned close to the image plane here represented by the camera sensor, the +1st diffraction order is minimally elongated compared to the point spread function of the 0th order and can therefore be analysed using common subpixel single-molecule localization algorithms. The distance between both PSFs provides accurate information on the spectral properties of the emitter. The minimal excess width of 1st order PSFs enables a fivefold higher emitter density compared to other sSMLM approaches whilst achieving a spatio-spectral localization accuracy sufficient to discriminate between fluorophores whose peak emission are less than 15 nm apart as demonstrated using dSTORM, DNA-PAINT and smFRET. We provide an ImageJ/Fiji plugin (sSMLMAnalyzer) and suitable Matlab scripts for data analysis. We envision that our approach will find widespread use in super-resolution applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

Pre-print: Multi-scale imaging of protein oxidation in mayonnaise

S. Yang, M. Takeuchi, H. Friedrich, J.P.M. van Duynhoven, and J. Hohlbein, 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 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 spintrap and protein autofluorescence. The effect of variation of pH and addition of EDTA on accumulation of spintraps validated that protein radicals were induced by lipid radicals. We observed protein radical formation at both the oil/water droplet interface and in the continuous phase. Our data suggests two main pathways of oxidative protein radical formation in LDL particles: at the droplet interface, induced by lipid free radicals formed in oil droplets, and in the continuous phase induced by an independent LDL-specific mechanism.

Published: Enabling single-molecule localization microscopy in turbid food emulsions

A. Jabermoradi, S. Yang, M. Gobes, J.P.M. van Duynhoven, and J. Hohlbein, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 380, 20200164, 2022,  [link], preprint on bioRxiv: [link]

Turbidity poses a major challenge for the microscopic characterization of food systems. Local mismatches in refractive indices, for example, lead to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, we added adaptive optics (AO) and flat-field illumination to our previously published open microscopy framework, the miCube. In the detection path, we implemented AO via a deformable mirror to compensate aberrations and to modulate the emission wavefront enabling the engineering of point spread functions (PSFs) for single-molecule localization microscopy (SMLM) in three dimensions. As a model system for a non-transparent food colloid such as mayonnaise, we designed an oil-in-water emulsion containing the ferric ion binding protein phosvitin commonly present in egg yolk. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain two- and three-dimensional images of phosvitin covered oil droplets with sub 100 nm resolution. Our data indicated that phosvitin is homogeneously distributed at the interface. With the possibility to obtain super-resolved images in depth, our work paves the way for localizing biomacromolecules at heterogeneous colloidal interfaces in food emulsions.

This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.

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

J. Hohlbein, Food Structure, 30, 100236, 2021, [link]

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.

Pre-print: Open microscopy in the life sciences: Quo Vadis?

J. Hohlbein, B. Diederich, B. Marsikova, E.G. Reynaud, S. Holden, W. Jahr, R. Haase, and K. Prakash, arXiv, 2021, [link]

Light microscopy allows observing cellular features and objects with sub-micrometer resolution. As such, light microscopy has been playing a fundamental role in the life sciences for more than a hundred years. Fueled by the availability of mass-produced electronics and hardware, publicly shared documentation and building instructions, open-source software, wide access to rapid prototyping and 3D printing, and the enthusiasm of contributors and users involved, the concept of open microscopy has been gaining incredible momentum, bringing new sophisticated tools to an expanding user base. Here, we will first discuss the ideas behind open science and open microscopy before highlighting recent projects and developments in open microscopy. We argue that the availability of well-designed open hardware and software solutions targeting broad user groups or even non-experts, will increasingly be relevant to cope with the increasing complexity of cutting-edge imaging technologies. We will then extensively discuss the current and future challenges of open microscopy.

Published: Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework

K.J.A. Martens, A. Jabermoradi, S. Yang, and J. Hohlbein, Methods, 193, 2021, [link], previously on bioRxiv [link]

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub- pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.

News: New lab members

For the start of the academic year, we welcome new lab members: Erwin Dijkstra started with his MSc thesis on spectrally resolved super-resolution imaging, Victor Pools will help us as a research assistant with cloning and single-molecule particle tracking, and Konstantin Speckner is taking a short break from his PhD in Bayreuth to learn more about single-molecule and single-cell techniques. Welcome on board!

Published: Probing DNA – transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices

M. Fontana, Š. Ivanovaite , S. Lindhoud, E. van der Wijk, K. Mathwig, W. van den Berg, D. Weijers, and J. Hohlbein, Advanced Biology, 2100953, 2021, [link], preprint: bioRxiv, 2021, [link]

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

News: Presentation on open microscopy and in-vivo single-molecule CRISPR-Cas

In January 2021, I had the pleasure to present some of our recent work as part of the Imaging ONEWORLD series initiated by the Royal Society of Microscopy. Topics I talked about include: open-source microscopy (#miCube), accelerated single-molecule localisation analysis (#SMLM) using phasor analysis, diffusion distribution analysis (#anaDDA), and in vivo single-particle tracking of CRISPR-Cas9.

News: NWO Take-off (phase I) granted & new lab members

Happy to share the news that we received a NWO Take-off (phase 1) grant for “Spectrally-resolved single-molecule localization microscopy to go”. Looking forward working with Niels and the people from Cairn Research Ltd, Confocal.nl, Wageningen University & Research on this exciting project to bring more colours into microscopy. 

We further say good bye to Mattia, who will continue working with my new colleague Dr. Sonja Schmid (see her new and shiny webpage here). Hope we can get all those great work of yours published soon!

We also welcome two new BSc students, Mink Neeleman and Casper Peters, who will work on CRISPR-Cas, live cell imaging and microfluidics. Welcome on board!