Optimized Stochastic Optical Reconstruction Microscopy for Imaging Chromatin Structure in Pathological Tissue

Area of Science:

• Cell Biology
• Microscopy
• Genomics

Background:

• Direct visualization of higher-order chromatin structure is crucial for understanding gene expression.
• Pathological processes involve chromatin structure changes, necessitating analysis in clinical samples.
• Formalin-fixed, paraffin-embedded (FFPE) tissues are the standard for pathological sample preservation.

Purpose of the Study:

• To detail an optimized stochastic optical reconstruction microscopy (STORM) protocol, named PathSTORM.
• To enable high-quality super-resolution imaging of higher-order chromatin organization in pathological tissues.
• To provide methods for fluorescence staining and key technical factors for STORM imaging in FFPE samples.

Main Methods:

• Development of PathSTORM, an optimized STORM protocol.
• Detailed methods for fluorescence staining of DNA and histone proteins.
• Key technical considerations for STORM imaging in pathological tissue samples.

Main Results:

• Successful application of PathSTORM for super-resolution imaging of chromatin in FFPE tissues.
• High-quality visualization of densely packed higher-order chromatin organization.
• Demonstration of essential steps for fluorescence staining and STORM data processing.

Conclusions:

• PathSTORM is an effective protocol for visualizing chromatin structure in pathological FFPE tissues.
• The protocol facilitates research into the role of chromatin organization in disease.
• This method advances the study of molecular-scale chromatin organization in clinical samples.