BPS2026 – Expansion microscopy through tensile force

Studying biological systems at their native spatial and temporal scales is essential to gain useful insights into relevant biophysical phenomena. Super-resolution techniques exist to surpass the diffraction limit of light to probe structure at relevant spatial scales. Expensive and specialized hardware or heavy post processing is often required to achieve super-resolution imaging. Expansion Microscopy (ExM), a sample preparation method, facilitates super-resolution imaging by leveraging osmotic forces to physically separate structures smaller than the diffraction limit. However, fragmentation and deformation are common issues that are hard to control in osmotic ExM. Further, labeling and signal loss due to digestion and dilution, controllability of expansion, and time-consuming sample preparation and imaging, are areas of potential improvement. Here, we develop tensile ExM by employing highly stretchable double network alginate-acrylamide gels that expand with the application of tensile force using a homebuilt iris-based mechanical expansion device to obtain more controlled expansion with less distortion and, less fragmentation, and higher throughput. We incorporate a resin-based microscale fluorescent fiducial marker grid made using two-photon polymerization to quickly identify and locate samples of interest within the stretchable hydrogels. The robust markers do not experience signal loss due to digestion or expansion. During sample homogenization, we observe a ∼3× expansion of the gel through osmotic forces. Subsequently, our mechanically expandable gels expand up to 4× in a controllable and repeatable manner to effectively expand the sample to ∼12×. We demonstrate our tensile ExM method by imaging fixed 3T3 cells, using immunohistochemistry-stained antibodies to tag microtubules and calthrin coated pits, achieving a spatial resolution of up to ∼60 nm. Tensile ExM is also compatible with other analytical methods that are sensitive to the additional water required in traditional osmotic ExM methodologies.