DNA-PAINT Imaging with Hydrogel Imprinting and Clearing.

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Hydrogel embedding is a key technique in fluorescence microscopy for specimen stabilization and optical clearing.
• DNA-PAINT (Direct DNA-based ANchorAGE-PAINT) is a super-resolution technique relying on oligonucleotide diffusion and binding.
• The compatibility of DNA-PAINT with hydrogel-embedded samples remained unexplored.

Purpose of the Study:

• To investigate the feasibility of DNA-PAINT in hydrogel-embedded biological samples.
• To assess the impact of hydrogel embedding on oligonucleotide diffusion and binding dynamics.
• To evaluate the structural integrity of biological specimens after hydrogel embedding and processing.

Main Methods:

• Utilizing polyacrylamide hydrogels for embedding fixed cells and DNA origami nanostructures.
• Employing acrydite-anchored oligonucleotides for DNA-PAINT imaging.
• Applying protease treatment for sample clearing and assessing its effects.
• Analyzing nanoscale positioning of docking strands using super-resolution microscopy.

Main Results:

• Polyacrylamide hydrogels facilitate sufficient oligonucleotide diffusion for successful DNA-PAINT imaging.
• Hydrogel embedding preserves the nanoscale positioning of docking strands, as demonstrated with DNA origami and microtubules.
• Protease treatment minimally affected microtubule structure while enhancing diffusion and accessibility of docking strands.
• Successful super-resolution imaging of hydrogel-embedded samples was achieved.

Conclusions:

• Hydrogel embedding is compatible with diffusion-based super-resolution microscopy techniques like DNA-PAINT.
• This approach offers a promising method for advanced imaging of biological structures within hydrogels.
• Further applications in diffusion and binding-based fluorescence imaging are anticipated.