Molecule Differentiation Encoding Microscopy to Dissect Dense Biomolecules in Cellular Nanoenvironments Below Spatial Resolution

Cellular biomolecules may exhibit dense distribution and organization at the nanoscale to govern vital biological processes. However, it remains a common challenge to digitize the spatially dense biomolecules under spatial resolution of microscopies. Here, we report a proof‐of‐principle method, molecule differentiation encoding microscopy by orthogonal tandem repeat DNA identifiers, to resolve the copy numbers of dense biomolecules in cellular nanoenvironments. The method encodes each copy of same biomolecules into different types of DNA barcodes based on stochastic multiplexed reactions. It can transform the overlap of the same spectrum into the overlap of different spectra. Furthermore, an algorithm is developed to automatically quantitate overlapping spots and individual spots. Using this method, we dissected RNAs in the cytoplasm, DNA epigenetic modifications in the cell nucleus, and glycans and glycoRNAs on the cell surface, respectively. We found that all these biomolecules presented dense distribution with diverse degrees in crowded cellular nanoenvironments. Especially, averaged 17% copies of U1 glycoRNA of single cells are gathered in various nanoenvironments on cell surface. Our strategy provides a powerful tool for digitally quantitative visualization of dense biomolecules below spatial resolution of microscopies, and could provide insights into underlying functions and mechanisms of the dense distribution information.