Combining ultrastructure expansion microscopy with immunofluorescence and Oligopaint DNA FISH

Area of Science:

• Cell Biology
• Microscopy Techniques
• Genomics

Background:

• Expansion microscopy (ExM) provides nanoscale imaging of subcellular structures using standard light microscopes.
• ExM is an accessible alternative to super-resolution microscopy.
• Combining ExM with Oligopaint DNA fluorescence in situ hybridization (FISH) has not been previously reported.

Purpose of the Study:

• To develop and optimize an ExM workflow for simultaneous Oligopaint DNA FISH and immunofluorescence (IF) in intact Drosophila ovaries.
• To enable high-resolution studies of nuclear organization in Drosophila.

Main Methods:

• The optimized protocol involves embedding Drosophila ovaries in a swellable hydrogel.
• Key steps include nucleic-acid anchoring, protein retention, and controlled digestion.
• Simultaneous Oligopaint DNA FISH and IF were performed on expanded samples.

Main Results:

• The workflow achieved approximately 5x expansion of the Drosophila ovary samples.
• Strong signal retention was maintained throughout the expansion process.
• The method successfully preserved chromatin integrity while ensuring probe accessibility.

Conclusions:

• This optimized ExM workflow enables simultaneous Oligopaint DNA FISH and IF in Drosophila ovaries.
• The technique achieves high-resolution imaging of nuclear organization with preserved subcellular structures.
• This approach offers a powerful tool for nanoscale biological research.