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Related Experiment Video

Updated: May 12, 2025

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DNA-PAINT Imaging with Hydrogel Imprinting and Clearing.

Johannes Stein1,2, Lorenzo Magni1,2, George M Church1,2

  • 1Wyss Institute of Biologically Inspired Engineering, Boston, Massachusetts 02215, United States.

ACS Sensors
|May 9, 2025
PubMed
Summary
This summary is machine-generated.

Polyacrylamide hydrogels enable diffusion-based super-resolution microscopy, like DNA-PAINT, by preserving nanoscale structures and improving accessibility for advanced biological imaging.

Keywords:
DNA-PAINTHydrogel embeddingMatrix imprinting and clearingPolyacrylamide gelsSingle-molecule localization microscopySuper-resolution microscopy

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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.