Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy

Area of Science:

• Cell Biology
• Molecular Biology
• Bioimaging

Background:

• Fluorescence in situ hybridization (FISH) on three-dimensionally preserved cells (3D-FISH) analyzes DNA/RNA localization and arrangement.
• Confocal laser scanning microscopy (CLSM) reconstructions were the gold standard for 3D-FISH.
• Super-resolution microscopy offers enhanced resolution beyond the diffraction limit.

Purpose of the Study:

• To apply three-dimensional structured illumination microscopy (3D-SIM) for visualizing subnuclear 3D-FISH preparations.
• To highlight the advantages of 3D-SIM for analyzing spatial relations and substructures of nuclear targets.
• To provide optimized protocols for super-resolution 3D-FISH in mammalian cells.

Main Methods:

• Utilized three-dimensional structured illumination microscopy (3D-SIM) for super-resolution imaging.
• Performed fluorescence in situ hybridization (FISH) on three-dimensionally preserved cells (3D-FISH).
• Developed optimized protocols for DNA/RNA-FISH, including combinations with immunofluorescence staining (Immuno-FISH) and click chemistry for DNA replication labeling.

Main Results:

• 3D-SIM provides an eightfold improved volumetric resolution compared to conventional imaging.
• Simultaneous visualization of differently labeled target structures is achievable with 3D-SIM.
• Identified challenges and provided solutions for optimal sample preparation, labeling, and acquisition in super-resolution 3D-FISH.

Conclusions:

• 3D-SIM represents a new state-of-the-art for bioimaging, particularly for 3D-FISH analyses.
• The enhanced resolution of 3D-SIM allows visualization of nuclear targets previously undetectable by conventional light microscopy.
• Optimized protocols are crucial for successful super-resolution imaging of in situ hybridizations.