Avdelningen för biomedicinsk teknik

Cellular function is governed by the molecular organization and interactions at the nanoscale. In this talk I will demonstrate our recent developments for improved 3D single-molecule tracking of dynamics and super-resolution imaging of nanoscale structures throughout mammalian cells and showcase applications of our approaches for cellular imaging. First, I will describe soTILT3D, an imaging platform that consists of a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background, photobleaching, and the risk of photodamaging sensitive samples, together with a 3D nanoprinted microfluidic chip for environmental control and for reflection of the light sheet into the sample. By combining these approaches with point spread function (PSF) engineering for nanoscale localization of individual molecules in 3D; deep learning for analysis of overlapping emitters; active 3D stabilization for drift correction and long-term imaging; and Exchange-PAINT for sequential multi-target imaging without chromatic offsets, we showcase whole-cell multi-target 3D single-molecule super-resolution imaging with improved accuracy, precision, and imaging speed. Next, I will demonstrate a versatile multimodal illumination platform that integrates the optical sectioning capabilities of light sheet illumination with uniform, flat-field epi- and TIRF illumination, resulting in more precise and accurate quantitation of single-molecule data. Finally, I will discuss how novel long axial-range double-helix PSFs offer stitching-free, 3D super-resolution imaging of whole mammalian cells, simplifying the experimental and analysis procedures for obtaining volumetric nanoscale structural information. Furthermore, we show that deep learning-based analysis drastically improves the achievable imaging speed and resolution with these PSFs. These imaging approaches are versatile and can be utilized to study molecular dynamics, nanoscale structures, and molecular mechanisms to address a broad range of chemical, biological, and biomedical questions related to cellular function and pathogenesis.

Microfluidics has emerged as a powerful tool with widespread applications across various scientific disciplines, including life sciences and catalytic chemistry. However, most existing microfluidic devices are static, lacking the ability to dynamically adapt microchannel geometries in response to changing experimental conditions. Here, we present an approach that integrates responsive microactuators within static microfluidic channels to engineer a reconfigurable microfluidic system using two-photon polymerization direct laser writing.
Our method enables precise control over the shape and positioning of thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) microactuators within microfluidic channels. By tuning the temperature, we exploit the lower critical solution transition temperature (LCST) of pNIPAM, which undergoes shrinkage above 32 °C and expands back to its original size at room temperature (22 °C). To demonstrate the versatility of this approach, we fabricated a micropillar brush array with varying gap sizes, allowing selective capture of polystyrene beads under constant flow. Upon heating, the micropillars shrink, increasing the gap size and releasing the previously trapped beads. Additionally, we designed paired microgrippers to effectively capture and release single cells, proving the biocompatibility and efficacy of our system in handling differently sized cells. To further enhance local stimulation and control, we incorporated a bistable multi-responsive microactuator system composed of pNIPAM and a light-responsive azobenzene compound. Using two-photon direct laser writing, we fabricated various microactuator geometries and exposed them to temperature and UV-light variations to demonstrate their bistable nature.
Our approach establishes a novel path towards reconfigurable microfluidics, enabling real-time experimental adjustments. Beyond applications in particle and cell manipulation, our system can be used to dynamically control flow regimes and release selected cells under constant flow conditionsThis advancement paves the way for next-generation microfluidic platforms with precise and programmable control over fluidic and biological interactions.

Cells are the basic entities of biological systems. They have particular physical properties, which enable them to navigate their 3D physical environment and fulfill their biological functions. We investigate these physical – mechanical and optical – properties of living cells and tissues using novel photonics and biophysical tools to test their biological importance. Our ultimate goal is the transfer of our findings to medical application in the fields of improved diagnosis of diseases and novel therapeutic approaches.

In this project, we have used the rapid developments in protein design and quantitative and structural mass spectrometry to establish a framework for antibody-guided design of protein-based nanoparticle immunogens using group A streptoccous (GAS) as a model system. We first developed a mass spectrometry based systems antigen-omics and systems serology workflow to select antigens in a data-driven manner. With these antigens as a starting point, monoclonal antibodies directed against key antigens were developed to define protective epitopes. These epitopes are in the final step redesigned and used to produce immunogens that display one or several epitopes in one immunogen. The immunogens assemble from two protein components i) a fusion component that carry the epitope and ii) the assembly component that when mixed, form ~3MDa icosahedral protein nanoparticles.                     
We have used the workflow to produce a proof-of-concept monovalent self-assembling protein-based nanoparticle immunogen against Streptolysin O (SLO), a prominent pore-forming toxin produced by GAS. For this immunogen, integrated structural mass spectrometry techniques and deep learning approaches were combined to re-engineer a protective epitope (D3m) present in domain 3 of SLO. D3m was displayed on the surface of a self-assembling icosahedral nanoparticle (D3m-NP) to enhance epitope presentation and immunogenicity. Immunising mice with D3m-NP, induced a robust and epitope-focused antibody response leading to improved protection against a lethal GAS challenge compared to immunisation with a detoxified full-length SLO vaccine construct. The approach can be extended to other antigens to explore the possibility of developing multivalent protein-based nanoparticle immunogens against GAS and other bacterial pathogens.