Abstract
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. Expansion microscopy (ExM) is an attractive option to achieve such imaging; however, simultaneous ExM imaging of proteins and mRNAs has not been demonstrated. Here, a technique for simultaneous ExM imaging of proteins and mRNAs in cultured cells and tissue slices, which we termed dual-expansion microscopy (dual-ExM), is demonstrated. First, we verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. Dual-ExM is a versatile tool to study complex biological systems, such as the brain or tumor microenvironments, at a nanoscale resolution.
Highlights
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems
We defined two procedures: first, mRNAs of cultured cells were labeled with fluorescence in-situ hybridization (FISH) probes, and subsequently, proteins of the same cells were labeled with antibodies, a process we termed FISH-IF; second, proteins of cells were labeled with antibodies, and mRNAs of the same cells were labeled with FISH probes, a process we termed IF-FISH
We found that FISH labeling of mRNAs followed by the IF labeling of proteins induces the successful visualization of both molecular species after expansion
Summary
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. We verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. We combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. We demonstrate the simultaneous ExM imaging of both proteins and mRNAs in cultured cells and tissue slices through the optimization of a labeling procedure and a quantitative study on the degree of retention. We show that the labeling and ExM process demonstrated here, termed dual-ExM, can visualize both the proteins and mRNAs from the same specimens at a lateral resolution beyond the diffraction limit of light
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