Heat denaturation enables multicolor X10-STED microscopy.

Area of Science:

• Biotechnology
• Microscopy
• Cell Biology

Background:

• Expansion microscopy (ExM) enhances imaging resolution by physically enlarging specimens.
• Achieving high expansion factors often leads to reduced fluorescence intensity, limiting super-resolution capabilities.
• Existing methods struggle to balance large expansion with sufficient signal for detailed nanoscale imaging.

Purpose of the Study:

• To develop a novel protocol for significant sample expansion while maintaining or improving fluorescence signal.
• To enable nanoscale imaging resolution in biological samples using super-resolution microscopy.
• To overcome the limitations of dimming associated with high-expansion ExM.

Main Methods:

• Developed a high-temperature homogenization (X10ht) protocol for a single-step sample expansion up to 10-fold.
• Compared fluorescence intensity of X10ht-processed gels with those from enzymatic digestion (proteinase K).
• Applied multicolor stimulated emission depletion (STED) microscopy to expanded neuronal cell cultures and isolated vesicles.

Main Results:

• X10ht achieved up to 10-fold expansion in a single step, significantly increasing fluorescence intensity compared to enzymatic methods.
• Enabled nanoscale imaging with 6-8 nm resolution in neuronal cultures and vesicles using STED microscopy.
• Successfully expanded thicker brain samples (100-200 µm) up to 6-fold, preserving epitope integrity.

Conclusions:

• High-temperature homogenization (X10ht) is a robust method for achieving high expansion factors and improved fluorescence.
• X10ht facilitates nanoscale resolution imaging with super-resolution microscopy, overcoming previous limitations.
• The protocol supports advanced labeling strategies, including nanobodies and signal amplification, for enhanced biological insights.