Expansion microscopy with trypsin digestion and tyramide signal amplification (TT-ExM) for protein and lipid staining

Abstract

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers two major drawbacks, namely proteolysis and swelling effects that, respectively, induce protein loss and dilute fluorescence signals. Here, we report two improvements to expansion microscopy that overcome these two challenges, i.e., deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. Notably, we demonstrate better protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures following 2-h trypsin treatment. Subsequent lipid staining revealed the complex 3D membrane structures in entire cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. Thus, TT-ExM significantly enhances fluorescent signals and generates high-quality and ultrafine-resolution images under confocal microscopy.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus

“…Finally, we conducted super-resolution imaging using expansion microscopy to further characterize associations among KLHL17, SYNPO, and ER, as well as the organization of the spine apparatus. To do so, we modified a published protocol [40] by replacing 2 reagents [41]. First, we replaced an oligonucleotide-linked secondary antibody with a conventional fluorophoreconjugated secondary antibody.…”

“…First, we replaced an oligonucleotide-linked secondary antibody with a conventional fluorophoreconjugated secondary antibody. Second, we substituted proteinase K with trypsin to digest post-stained neurons embedded in a hydrogel [41]. Proteinase K is a potent and broad-spectrum protease that cleaves peptide bonds at the carboxylic terminal of aromatic, aliphatic, or hydrophobic amino acids.…”

“…Immunostained neurons, prepared as described above, were subjected to expansion microscopy as described previously [41], except that tyramide signal amplification was not applied. Briefly, the stained cells were incubated with 0.1 mg/ml Acryloyl-X, SE in PBS overnight at 4˚C.…”

Proximity Labeling Expansion Microscopy (PL-ExM) resolves structure of the interactome

“…PL-ExM is compatible with any PL methods that can biotinylated proteins, for example, APEX and HRP labeling. Interestingly, HRP-catalyzed tyramide signal amplification (TSA) was recently used to amplify signals for ExM 30 , but not for interactome visualization. PL-ExM was designed and optimized for the opposite purpose, that is proteome characterization.…”