Super-resolution co-imaging of proteins and nucleic acids on expansion microscopy

Visualization of proteins and nucleic acids with super-resolution is a persistent need. Expansion microscopy (ExM) permits nanoscale imaging of biomolecules on a conventional microscope by physically expanding biological specimens embedded in a stretchable hydrogel. However, achieving simultaneous super-resolution co-imaging of proteins and nucleic acids on ExM has remained a general challenge. Here, we present photoclick dual anchoring expansion microscopy (Phan-ExM), which employs an unorthodox anchoring reagent, <i>N</i>-(3-methacrylamidopropyl)-3-(2-methyl-1<i>H</i>-pyrrol-1-yl)-2<i>H</i>-tetrazole-2-carboxamide (MAP-mPyTC), for the rapid and concurrent retention of proteins and nucleic acids within the ExM gel matrix through photoclick chemistry. We demonstrated that MAP-mPyTC could anchor both protein and mRNA biomolecules within 10 min, significantly reducing the time compared to typical ExM techniques, which usually take from several hours to overnight. Moreover, we showed that Phan-ExM significantly enhanced the fluorescence intensity of protein and RNA spots compared to previous methods. With Phan-ExM, we achieved high-resolution co-imaging of multiplex proteins and nucleic acids on a single specimen at ∼85 nm resolution. We revealed that paclitaxel and colchicine treatment significantly disrupted mitochondrial dynamics in BALB/c3T3 cells, with an associated aggregation of ACTB mRNA observed at sites of mitochondrial damage. Phan-ExM is a platform technique that enables super-resolution co-localization of nucleic acids and proteins on the same specimen using a conventional microscope.