Jupiter hell igg11/5/2022 Second, superresolution microscopy often requires certain dyes that do not survive hydrogel polymerization. First, ensuring isotropic expansion at the nanometer scale requires more thorough proteinase digestion or denaturation hence, more labeled antibodies are washed out ( Chen et al., 2015 Chozinski et al., 2016 Tillberg et al., 2016). The label-loss problem is exacerbated when aiming for higher spatial resolution. Consequently, >50% of the target molecules can lose labeling after expansion ( Tillberg et al., 2016 Truckenbrodt et al., 2019), which is a major issue of current ExM methods. #JUPITER HELL IGG FREE#Nevertheless, digestion and denaturation cause incompletely anchored proteins or protein fragments to be washed out ( Chozinski et al., 2016 Chung et al., 2013 Yang et al., 2014), the polymerization to make the hydrogel produces free radicals that readily destroy fluorophores ( Chen et al., 2015 Tillberg et al., 2016 Truckenbrodt et al., 2019), and both factors can damage fluorescent proteins. To retain the spatial information of the target structures, the biomolecules of interest, such as protein ( Chozinski et al., 2016 Ku et al., 2016 Tillberg et al., 2016) or RNA ( Chen et al., 2016), and/or labels, such as dye-labeled DNA ( Chen et al., 2015), dye-labeled antibodies ( Chozinski et al., 2016 Tillberg et al., 2016), or fluorescent proteins ( Ku et al., 2016 Tillberg et al., 2016), are anchored to the hydrogel matrix before digestion or denaturation. In all of these cases, the homogenization of the specimen through either protease digestion ( Chen et al., 2015 Chozinski et al., 2016 Tillberg et al., 2016) or protein denaturation ( Ku et al., 2016 Tillberg et al., 2016) is essential to achieve isotropic expansion without structural distortion. Recent efforts have further enhanced the resolution of ExM either by increasing the volume expansion ratio ( Chang et al., 2017 M’Saad and Bewersdorf, 2020 Truckenbrodt et al., 2018) or by combining ExM with superresolution microscopy, such as structured illumination microscopy (SIM Cahoon et al., 2017 Halpern et al., 2017 Wang et al., 2018) and stimulated emission depletion (STED) microscopy ( Gambarotto et al., 2019 Gao et al., 2018 Li et al., 2018 Xu et al., 2019). By physically expanding the sample before image acquisition, expansion microscopy (ExM) has enabled the use of a conventional confocal microscope to achieve ∼70-nm lateral spatial resolution ( Chen et al., 2015 Chozinski et al., 2016 Ku et al., 2016 Tillberg et al., 2016).
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