Multiplexing spatially resolved omics with increased resolution: Development of an expansion microscopy platform

Cells are the fundamental functional units in all forms of life. Even though largely all cells in a human body carry the same genetic information, we still know surprisingly little about their detailed composition. This is due to the vast amount of variation in a cell's gene expression. Specifically, each cell at a certain moment in time has a certain set of genes that are switched on or off, resulting in different biomolecules which are being expressed, in their turn regulating cellular processes. In order to understand the biological driving forces of health and disease, a detailed map of the trillions of different cells with their specific biomolecules and within their natural environment is needed. However, as biomolecules are often much smaller than 200 nm, direct visualization in large quantities, e.g. through light microscopy imaging, is challenging. Recently, a method named expansion microscopy addresses this issue in an elegant and cost-effective way. Compared to other, more classical super-resolution fluorescence microscopy techniques, expansion microscopy embeds biological samples in a water-absorbing polymer which can be physically expanded, laying bare their subcellular structures with nanometer precision on conventional light microscopes. Following this approach, expansion microscopy has several advantages over other super-resolution fluorescence microscopy methods. Yielding a perfectly transparent matrix, it is most suitable for nanoscale imaging of volumetric samples such as pieces of tissue. For this reason, the field is now rapidly evolving in a direction where it is combined with state-of-the-art fluorescent methods for single cell genome, transcriptome and proteome mapping in multicellular samples. Nevertheless, expansion microscopy is still in its infancy, and certain hurdles remain to be tackled. In this thesis, we aim to contribute to this endeavor, evaluating whether ExM is a suitable platform used to multiplex different omics, by studying if expansion is isotropic, and if heterogeneities in sample composition influence the expansion factor. We found that the expansion hydrogel indeed has the potential to ensure isotropic expansion in all dimensions, but care should be taken during sample handling. Furthermore, as cell function is determined by different DNA, RNA and protein markers, the next step will be to study a combination of biomarkers in their spatial context via integrative techniques. For this reason, we seek for improved labeling strategies viii which better retain fluorescent signal in the expanded sample, and which facilitate simultaneous crosslinking of different biomolecules. These efforts have resulted in an optimized expansion microscopy workflow, which was adopted to perform the first tests for implementing a protein multiplexing readout-scheme in expansion microscopy, referred to as immuno-SABER. Finally, we explored which resolution can be achieved when imaging expanded samples on superresolution microscopy setups, with possible applications in resolving protein ultrastructures with nanoscale precision. Overall, this work brought us one step closer to using expansion microscopy as a multi-modal fluorescent detection platform with higher efficiency and reliability.

Publication year:2021

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