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Expansion   Microscopy

About ExM

For many years now, fluorescence microscopy has established itself as an indispensable tool for numerous biologists. However, the physical laws governing light diffraction have restricted our view to length scales well above 250 nm. As the biomolecular actors which regulate all the fundamental processes of life are typically in the few nanometer range, our ability to gain true insights into many biological systems is severely compromised. Fortunately, continuous advancements in optics, electronics and mathematics have provided scientist with different so called ‘super resolution microscopy’ approaches which can overcome this diffraction barrier. A more recent approach called expansion microscopy obtains a higher resolution by physically expanding the sample rather than monitoring the microscope or the acquired data. An expandable polymer gel is introduced inside the biological sample, pulling apart individual biomolecules which results in resolutions of up to 10x smaller than normal on diffraction limited microscopes.




Expansion microscopy has several advantages over other super-resolution fluorescence microscopy methods. Yielding a perfectly transparent matrix in which the original structure of the samples is preserved, it is most suitable for nanoscale imaging of large volume samples, e.g. tissue sections. Low penetration depth, dedicated objectives with short working distances or computational time make this not feasible for the more classical approaches. In an age where state-of-the-art methods for genome, transcriptome and proteome analysis are well established for single cell analysis, studying these single-cell ‘omics’ in their original environment becomes the next challenge.


While expansion microscopy has shown its potential for accomplishing this laborious task by imaging the distribution different RNA molecules or proteins in whole tissue sections, the technique is still in its infancy. Problems such as signal loss due to poor crosslinking need to be solved, and integrative techniques rather than full coverage techniques which measure only one type of molecule are necessary. In this effort, the Hofkens Lab is working towards an approach that allows for the simultaneous readout of different cellular biomarkers, while better crosslinking individual molecules to the polymer matrix. We are expanding the already existing toolbox with new, ExM-compatible labeling strategies to visualize the subcellular location of biomolecules in the context of more complex tissues.

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Key publications:

Chen, F.; Tillberg, P. W.; Boyden, E. S. Expansion Microscopy. Science 2015, 347, 543–548. 10.1126/science.1260088

Wen, G.; Vanheusden, M.; Acke, A.; Valli, D.; Neely, K. R.; Leen, V.; Hofkens, J. Evaluation of direct grafting strategies in Expansion Microscopy. BioRxiv 2019. DOI: 10.1101/696039

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