The investigation of complex biological systems with single molecule spatial precision and sensitivity has been a long-sought goal in biology.1 Recently we have developed an imaging method, termed expansion microscopy (ExM), capable of nanoscale imaging of cells and tissues in 3-D by isotropically expanding structures cross-linked into polyacrylate polymers.2 The expansion is achieved by anchoring fluorescent tags associated with biomolecules to polymer chains and mechanically homogenizing the original biological structure prior to the isotropic swelling in water. We discovered that the effective magnification resulting from the mechanical separation of fluorophores allows for optical imaging of nanoscopic features beyond diffraction limit. However, as yet the nanoscopic structure and morphology of the gels remains largely unexplored. Here, we report on the nanoscopic characterization of the polyacrylate polymers themselves. In particular, we analyze the polymer networks with nanoscopic imaging techniques such as in-situ atomic force microscopy (in-situ AFM) and cryo-scanning electron microcopy (cryoSEM). We describe a strategy and procedure to image the gel in its native or cryogenic states, in order to avoid major disturbances of the structure and morphology. Based on the direct observation of the polymer network, we report a systematic study of its surface morphology, pore size distribution and cross-linking density in different expansion states. Further, we show a direct observation of major morphological changes of the gel under different environments including the pH and ionic strengths. Finally, we carry out an in-situ dynamic study of the gel in order to elucidate the nanoscopic description and mechanism of the polymer network expansion in ExM. 1. Alivisatos, A. P, et al. ACS Nano 2013, 7, 1850-1866. 2. Chen, F.; Tillberg, T. W.; Boyden, E. S. Science 2015, 347, 543-548.

Expansion microscopy: Nanoscopic characterization of polyacrylate polymers

251st National Meeting (2016)

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