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Interested in quantitative (super-resolution) microscopy, working at the interface of optics and cell biology, and shaping the future of a young research group? We are always looking for talented and motivated people to join our team.
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Amyloid polymorphism
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Smart microscopy
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SOLEIL-SOFI
Gentle super-resolution imaging for tissue
publications
Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy
Abstract
Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going ‘beyond the diffraction barrier’ comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.
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Abstract
Parameter-free image resolution estimation based on decorrelation analysis
Abstract
Super-resolution microscopy opened diverse new avenues of research by overcoming the resolution limit imposed by diffraction. Exploitation of the fluorescent emission of individual fluorophores made it possible to reveal structures beyond the diffraction limit. To accurately determine the resolution achieved during imaging is challenging with existing metrics. Here, we propose a method for assessing the resolution of individual super-resolved images based on image partial phase autocorrelation. The algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in super-resolution microscopy.
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SOLEIL: single-objective lens inclined light sheet localization microscopy
Abstract
High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination.
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Mapping volumes to planes: Camera-based strategies for snapshot volumetric microscopy
Abstract
Optical microscopes allow us to study highly dynamic events from the molecular scale up to the whole animal level. However, conventional three-dimensional microscopy architectures face an inherent tradeoff between spatial resolution, imaging volume, light exposure and time required to record a single frame. Many biological processes, such as calcium signalling in the brain or transient enzymatic events, occur in temporal and spatial dimensions that cannot be captured by the iterative scanning of multiple focal planes. Snapshot volumetric imaging maintains the spatio-temporal context of such processes during image acquisition by mapping axial information to one or multiple cameras. This review introduces major methods of camera-based single frame volumetric imaging: so-called multiplane, multifocus, and light field microscopy. For each method, we discuss, amongst other topics, the theoretical framework; tendency towards optical aberrations; light efficiency; applicable wavelength range; robustness/complexity of hardware and analysis; and compatibility with different imaging modalities, and provide an overview of applications in biological research.
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research
Smart microscopy
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Publications:
Labelfree & quantitative phase imaging
Label-free optical methods such as quantitative phase imaging (QPI) can be used to access mechanical properties and changes in (intra-)cellular morphology non-invasively. The measurements provide information about the local thickness and refractive index at high imaging speed. This allows e.g. long-term monitoring of cell dry mass, enabling measurements of cell growth rate and mass transport.
Multiplane
Cells are three-dimensional objects and intracellular motion is generally not constrained to a single plane. Multiplane microscopy allows visualization of cellular structures and dynamics through the simultaneous imaging of different focal planes within the sample. We realize multi-plane microscopy using image splitting prisms for ultrafast (ms), inter-plane drift-free 3D data acquisition. This implementation allows imaging across the visible spectrum with high trans-mission efficiency. Prism-based imaging is compatible with different microscopy modalities from conventional (multicolor) fluorescence, single-particle tracking and super-resolution mi-croscopy (SOFI, SIM) to label-free quantitative phase imaging. We are developing and apply-ing open-source multiplane imaging hardware and software to facilitate widespread adoption.
Probes
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Neurodegeneration
Although the genetic origin of a multitude of neurodegenerative disease has been identified for decades, the underlying mechanism of protein aggregation remains poorly understood. With the advance and distribution of EM-methods, emphasis has been given to structural insights of protein aggregates and mutant substructure characterisation, thereby losing the temporal context of actual dynamics. Quantitative light microscopy methods can incorporate these insights to unveil structure-function relationships relevant to identify molecular interactions in the aggregation processes. Intracellular phase transitions, as a paradigm of misregulated protein self-assembly and increasing reports of proteins physiologically undergoing liquid-liquid phase separation, are a promising framework to investigate these neuropathologies.
Huntingtin (Htt) is our aggregation protein model system, which is linked to Huntington’s disease, a neurodegenerative disorder characterized by a progressive loss of striatal and cortical neurons. The pathology is thought to be caused by an expanded, unstable trinucleotide (CAG) repeat in the first exon (HttEx1) of the Htt gene, which translates to a polyglutamine (polyQ) repeat in the protein product. A multitude of potential and partially reversible aggregation pathways via soluble dimers and oligomers to protofibrils and amyloid fibers culminating in μm-scale inclusion bodies (IBs) have been proposed, in addition to various potential cytotoxic effects on neurons. The actual mechanism leading to aggregation, e.g. via chaperones, intrinsic aggregation due to misfolding and PTMs, or cellular modulators remains to be determined. Steady progress has been made in uncovering disease mechanisms downstream of mHtt expression. For example, there is some consensus that polyglutamine-expanded mHtt elicits gain-of-function proteinopathy that may affect both nuclear and cytoplasmic cellular function.
Single molecule localisation
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SOFI
Team members:
Collaborators:
Publications:
 | Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy Adrien Descloux, Kristin Grussmayer, E. Bostan, T. Lukes, A. Bouwens, A. Sharipov1, S. Geissbuehler, A.-L. Mahul-Mellier, H. A. Lashuel, M. Leutenegger and T. Lasser Published in Nature Methods, February 2018 (see publication) Research themes: SOFI Type: Paper |