Simon Sprecher¹
1. Department of Biology, University of Bern

Understanding the logic of how the brain is wired remains a challenge with hundreds of distinct cell types, that are interconnected in complex networks. Combining genetic techniques with single-cell transcriptomic approaches and imaging techniques provides a powerful approach to identify basic rules of how the brain is build and how information is encoded and processed. First, to investigate fundamental conding principles we use the powerful genetics of the fruit fly as powerful approach to express genetic coded Calcium sensors in sensory neurons as model to depict the capacity of sensory systems. Second, to understand how highly advanced brains can evolve independently we investigate the cephalopod brain by combining HCR to map gene expression with whole brain imaging. These two experimetnal paradigms provide an example of how integrating genetic, single-cell transcriptomics and state-of-the-art imaging techniques allow us to understand the basic functions of the brain.

Anja E. Hauser¹
1. Charité, Universitätsmedizin Berlin and Deutsches Rheuma-Forschungszentrum Berlin, Germany

My lab is interested in analyzing immune cells in the tissue context in health and disease. We aim to understand how the tissue microenvironment affects the metabolism of myeloid cells in the bone marrow, and how that impacts on their function. In order to analyze this process in 3D, we developed a tissue clearing, staining and light sheet fluorescence microscopy imaging pipeline called MarShie, optimized to image the entire intact femur at subcellular resolution down to the deepest bone marrow regions. We show that in aged mice the draining sinus massively decreases in volume while transcortical vessels also decline. During homeostasis CX3CR1+ myeloid cells localize in perivascular niches, whereas CD169+ myeloid cells are dispersed in the parenchyma. After injury, CX3CR1+ myeloid cells relocate and sequester the injury site prior to vascularization, acting as trailblazers for osteogenic type H vessels.

Phenotypes and functions of immune cells are linked to their metabolic profile. We developed longitudinal intravital imaging of the mouse femur, to enable micro-endoscopic fluorescence lifetime imaging (FLIM) for metabolic profiling (“MetaFLIMB”). Myeloid cells display highly heterogeneous metabolic profiles both spatially and temporally during bone regeneration, beyond the binary paradigm of myeloid cells using either glycolytic or oxidative signaling pathways linked to pro- or anti-inflammatory functions, respectively. Under in vivo conditions, myeloid cells with various metabolic profiles, i.e. using other pathways for energy production than the anaerobic pathway associated with pro-inflammatory functions, performed the oxidative burst necessary for the process of phagocytosis. This demonstrates that a high metabolic flexibility of myeloid cells in vivo is related to their functional flexibility.

Christian Soeller¹
1. Institut für Physiologie, University of Bern

MINFLUX microscopy is a second-generation optical super-resolution technique capable of achieving near-isotropic nanometre localization precision in all three dimensions [1]. This is typically achieved through rapidly scanning a bottle-beam shaped laser spot (AKA “3D doughnut”) over the sample and making single molecule localizations progressively closer to the zero position of the beam. Single-molecule events for MINFLUX microscopy can be induced through photo-activation/uncaging, photo-switching, or through transient binding of dye labelled small molecules.
We have established a MINFLUX facility around a commercial MINFLUX microscope at the Institute für Physiologie in Bern and have implemented routine molecular resolution imaging over the past year. This involved fully characterising the capabilities of the microscope and the resolution achievable in practice. At resolutions approaching the single nanometre scale drift and any related processes that can degrade the image resolution need to be carefully compensated and monitored. We have used synthetic DNA origami structures to measure the in-situ performance of the microscope and established routines to allow nanometre precision imaging over several hours.
We demonstrate the utility of MINFLUX for imaging of receptor distributions in biological cells using the cardiac ryanodine receptor (RyR), a large 2 MD homo-tetramer that acts as intracellular calcium channel in heart cells [2]. We use single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions (RyR2-GFP in HEK293 cells and RyR-PA-tagRFP in myocytes from a PA-RFP RyR2 knock-in mouse line) to determine the location of individual RyR2 subunits with high precision (~3 nm) in all directions.
Super-resolution imaging with MINFLUX also offers the ability to measure the effective labelling efficiency in the sample. Labelling efficiency is rarely measured in biological experiments but is a critical parameter that can vary over more than an order of magnitude depending on the preparation. To estimate the effective in-situ labelling efficiency we have used side-by-side 3D MINFLUX imaging of nuclear pore complexes containing NUP96-eGFP and cells expressing RyR2-GFP. Cross-calibration showed that with optimisation labelling efficiency reaches 60% and better with refined MINFLUX imaging protocols. For the large (~27 nm) RyR-tagRFP homo-tetramers this allowed not only estimating the location at high precision but also the 3D orientation of individual RyR2 channels in intact cells.
Our data shows that MINFLUX microscopy can provide information on 3D protein orientation in the cell that was previously only available with electron microscopy. In addition, it has the scope to enable molecular tracking in cells with high temporal and spatial resolution.

[1] Balzarotti et al., Nanometer resolution imagining and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606-612 (2017). DOI: 10.1126/science.aak9913
[2] Clowsley, A. H., et al. Analysis of RyR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D MINFLUX microscopy. biorXiv 2023.07.26.550636

Mario Prsa¹

1. Department of Neuroscience and Movement Science, University of Fribourg, Switzerland.

The act of moving is both a motor and sensory process. To displace a limb along a desired trajectory, position signals sent from the limb back to the brain exert feedback control by shaping the motor command during execution. This sensation of where the limb is located in space is called proprioception (the often overlooked ‘sixth sense’) and is indispensable for moving accurately. Proprioception is a highly understudied sensory modality and one of the main unsolved puzzles of sensorimotor neuroscience. We know very little about the underlying brain circuits, how neurons encode proprioceptive information or how it is consciously perceived. These questions have been difficult to address because proprioception cannot be manipulated experimentally with the same level of precision as other sensory modalities. To solve this challenge we developed a robotic system for delivering precisely quantifiable proprioceptive stimuli to the mouse forelimb while simultaneously imaging and manipulating in vivo neuronal activity in the cortex and cerebellum with state-of-the-art microscopy and genetic tools. Our findings suggest that the proprioceptive neural code represents spatial variables that interface the limb with the body’s peripersonal space.

Aleksandra Radenovic,¹

1. Institute of Bioengineering, School of Engineering, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland

In this talk, I will demonstrate how advancements in imaging methods can open new avenues for research at solid-liquid interface. In the first part, I will discuss our efforts to explore nanophotonics at the single-molecule level, with a focus on the optical dynamics and conformations of individual emitters at the solid-liquid interface and in confined environments.
In our recent work, we employed fluorescence microscopy to monitor electrochemical reactions at the single-molecule scale, achieving a wide field of view and high temporal resolution. Additionally, we introduced an advanced bifocal polarization single-molecule localization microscopy (pSMLM) to enable real-time, multi-dimensional observations of quantum emissions formed by organic molecules and h-BN native defects. Our findings reveal a strong correlation between the orientation of quantum emitters and the symmetry of the h-BN lattice.
I will also discuss the use of SPAD cameras for single-particle tracking applications, as well as in high-throughput single-molecule fluorescence lifetime imaging (smFLIM).

[1]. Ronceray, Nathan, Yi You, Evgenii Glushkov, Martina Lihter, Benjamin Rehl, Tzu-Heng Chen, Gwang-Hyeon Nam et al. "Liquid-activated quantum emission from pristine hexagonal boron nitride for nanofluidic sensing." Nature Materials 22, no. 10 (2023): 1236-1242.
[2]. Mayner, Eveline, Nathan Ronceray, Martina Lihter, Tzu-Heng Chen, Kenji Watanabe, Takashi Taniguchi, and Aleksandra Radenovic. "Monitoring electrochemical dynamics through single-molecule imaging of hBN surface emitters in organic solvents." arXiv preprint arXiv:2405.10686 (2024).
[3]. Guo, Wei, Tzu-Heng Chen, Nathan Ronceray, Eveline Mayner, Kenji Watanabe, Takashi Taniguchi, and Aleksandra Radenovic. "Dipole orientation reveals single-molecule interactions and dynamics on 2D crystals." arXiv preprint arXiv:2408.01207 (2024).

Derek Kiebala,^1,3, Robert Style,^1,2 Dimitri Vanhecke,³ Celine Calvino,⁴ Christoph Weder,³ Stephen Schrettl,^3,5
1 Department of Chemistry, University of Mainz, 55130 Mainz, Germany
2 Department of Materials, ETH Zurich, 8093 Zurich, Switzerland
3 Adolphe Merkle Institute, University of Fribourg, 1700 Fribourg, Switzerland
4 University of Freiburg, 79110 Freiburg, Germany
5 TUM School of Life Sciences, Technical University of Munich, 85354 Munich, Germany

Blending conventional polymers with aggregachromic dyes is a convenient technique to fabricate mechanoresponsive luminescent (MRL) materials, but the approach is largely limited to semi-crystalline polymers.[1] To overcome this challenge, we developed a telechelic poly(ethylene-co-butylene) terminated with excimer-forming cyano-oligo(p-phenylene-vinylene) dyes (tOPV) as an additive that can render different types of polymers mechanochromic[2] and demonstrated the versatility of this tOPV additive as a sensitive, reversible strain sensor in commercial polyurethanes (PUs) with varying degrees of crystallinity.[3] Here, we use confocal laser scanning microscopy (CLSM) to elucidate the operating principles that govern the mechanochromism of tOPV.[4] CLSM images of polymer films blended with tOPV reveal the presence of emissive, spherical Eschelby inclusions that become elongated and change their fluorescence color when the sample is deformed (Figure 1). Moreover, in-situ monitoring of individual inclusions during deformation yields information on the inclusions’ stiffness, as well as the extent of tOPV aggregation within the inclusions as a function of applied strain. The mechanochromic inclusions and CLSM imaging techniques reported here constitute a new paradigm for visualizing the transfer of bulk forces from the macro- to the nanoscale, and we believe that the mechanistic insights presented herein will facilitate the development of new and effective MRL materials.

[1] H. Traeger, D. J. Kiebala, C. Weder, S. Schrettl, Macromol. Rapid Commun. 2021, 42, 2000573.
[2] C. Calvino, Y. Sagara, V. Buclin, A. P. Haehnel, A. del Prado, C. Aeby, Y. C. Simon, S. Schrettl, C. Weder, Macromol. Rapid Commun. 2019, 40, 1800705.
[3] D. J. Kiebala, Z. Fan, C. Calvino, L. Fehlmann, S. Schrettl, C. Weder, Org. Mater. 2020, 02, 313–322.
[4] D. J. Kiebala, R. Style, D. Vanhecke, C. Calvino, C. Weder, S. Schrettl, Adv. Funct. Mater. 2023, 33, 2370297.

Viola Bauernfeind,^1,2, Kenza Djeghi,^1,2 Vinodkumar Saranathan,³ Ullrich Steiner,^1,2 Bodo Wilts,^2,4
1 Adolphe Merkle Institute, University of Fribourg, Switzerland
2 National Centre of Competence in Research Bio-Inspired Materials, Switzerland
3 Division of Sciences, School of Interwoven Arts and Sciences, Krea University
4 Department Chemistry and Physics of Materials, Paris-Lodron University Salzburg, Austria

Color is ubiquitous in nature and arises from various interactions of light with biological materials. Absorption of light by dyes typically produces pigmentary coloration, while structural coloration arises from interference effects with materials structured on the length scale of the wavelength of light. Such photonic nanostructures take a variety of shapes and are widespread in the animal kingdom. For example, they color the wings of Morpho butterflies a radiant blue and give jewel beetles their glossy, multicolored appearance.
Recent research focuses on natural and synthetic disordered photonic structures that require advanced characterization techniques. Many photonic structures are porous, thus achieving high refractive index contrast and often good scattering contrast in scattering experiments. Porosity, however, poses another challenge for volume imaging in a slice-and-view approach because it comes with a finite depth of view. We advanced the quantification of disorder in porous three-dimensional photonic structures by in-situ platinum-backfilling of the porous structures for contrast-enhanced volumetric imaging using focused ion beam tomography. Using this technique, we studied the ultrastructural origins of vibrant color patterns in Sternotomini longhorn beetles that bear colored scales containing bicontinuous photonic networks. We combined focused ion beam tomography that allows for a statistical analysis of the network morphology based on network skeletonization with complementary ultra-small-angle X-ray scattering analyses. Our results suggest evolutionary relations between beetles with morphologically similar networks that differ in their extent of disorder and emphasize the need for further research to understand the formation of these complex three-dimensional photonic structures.

Guillermo Acuna,¹

1. Photonic Nanosystems, Department of Physics, University of Fribourg, Switzerland

Developments in low-cost microscopy have accelerated greatly in recent years due to the technological advances of modern smartphones. Among different features, these devices have image sensors with more pixels, better quantum efficiencies, better optics design for light collection, and larger focal distances in different lenses of multi-camera smartphones. Distinct aspects of smartphones, i.e., portability and compactness, have also pushed forward the development of specific smartphone-based setups useful in Point-Of-Care (POC) applications like clinical diagnostics, quantification of immunoassays, detection of bacteria, cancer cytology, fresh tissue imaging, lead and microplastics quantification. While most of these applications used optical setups designed for fluorescence imaging, only a few of them focused on the detection of single molecule fluorescence.
Here, we developed a portable and inexpensive smartphone-based fluorescence microscope that detects direct emission from single molecules. We tested its performance by analyzing single-molecule intensity traces with three smartphones. We also demonstrated that it can be used for super-resolution microscopy with a Single-Molecule Localization Microscopy (SMLM), DNA-PAINT. The smartphone-based microscope we present is low-cost, portable, easy to use, and can virtually be used with any smartphone, making an impact on a truly broad audience.

Pablo Rivera-Fuentes,¹
1 Laboratory of Chemical and Biological Probes, Department of Chemistry, University of Zürich, Switzerland.

Fluorophores with intermittent emission patterns, often called “blinking”, have been used for single-molecule imaging in super-resolution microscopy. In some cases, these blinking patterns emerge from a ground-state isomerization between a fluorescent and a non-fluorescent form of the dye. We observed that the blinking pattern of a spontaneously blinking dye was sensitive to its environment. In this presentation, I will show that the fluorescence intermittency of a peptide or protein labeled with a spontaneously blinking fluorophore contains information about the structure of the biomolecule. Using a deep learning algorithm, this single-molecule blinking pattern can be used to identify the peptide or protein without the need to sequence it. This method can distinguish between peptides with different sequences, peptides with the same sequence but different phosphorylation patterns, and peptides that differ only by the presence of epimerized residues. Proteins can also be identified with high accuracies, and two different labeling sites of the same protein can also be accurately detected. This study builds the foundation for a protein identification technique with single-molecule sensitivity.

Sophie Brasselet¹

1. Institut Fresnel, Aix Marseille Univ, CNRS, Centrale Med, Marseille, France

We report the imaging of the orientation of fluorescent single molecules in 3D, using polarized optical microscopy at high numerical apertures. We apply this approach to super resolution imaging of proteins’ organizations in cells and extend its capabilities to circularly polarized emission.