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Researchers can now perform deep, whole-cell single-molecule localization microscopy (SMLM) using standard spinning disk confocal microscopes. This breakthrough enables nanoscale imaging throughout the entire cell volume, expanding biological research possibilities.

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Area of Science:

  • Cellular and Molecular Imaging
  • Biophysics
  • Microscopy Techniques

Background:

  • Single-molecule localization microscopy (SMLM) offers nanoscale resolution but is typically limited to near-surface cellular imaging.
  • Conventional illumination methods like TIRF and HILO restrict SMLM to regions close to the coverslip.
  • There is a need for SMLM techniques that can image entire cells in three dimensions.

Purpose of the Study:

  • To present a protocol for performing whole-cell SMLM using a spinning disk confocal microscope with optical photon reassignment (SDC-OPR).
  • To enable high-precision single-molecule imaging throughout the full cell volume using DNA points accumulation for imaging in nanoscale topography (DNA-PAINT).
  • To provide an accessible method for deep SMLM using standard confocal equipment without custom optics.

Main Methods:

  • Utilized a spinning disk confocal microscope equipped with optical photon reassignment (SDC-OPR).
  • Employed DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with DNA-conjugated probes for intracellular labeling.
  • Developed stepwise guidance for microscope configuration, acquisition parameter optimization for deep imaging, and sample preparation for multicolor imaging.

Main Results:

  • Successfully demonstrated whole-cell SMLM using SDC-OPR and DNA-PAINT on standard confocal microscopes.
  • Achieved high-precision single-molecule imaging throughout the full cell volume.
  • Provided critical considerations for minimizing background, optimizing resolution, and ensuring imaging quality in deep cellular regions.

Conclusions:

  • This protocol enables deep, whole-cell SMLM using readily available confocal microscopy equipment.
  • The SDC-OPR method significantly expands the scope of biological questions addressable by SMLM beyond near-membrane regions.
  • Researchers can now explore nanoscale structures within the entire cell volume with high precision.