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We developed a new deep-tissue super-resolution microscope, confocal² spinning-disk image scanning microscopy (C²SD-ISM), to overcome background interference and improve imaging depth and fidelity for biological research.

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Area of Science:

  • Biophotonics and advanced imaging techniques.
  • Microscopy and super-resolution imaging.
  • Cellular and tissue imaging.

Background:

  • Deep-tissue super-resolution imaging is limited by background noise, reducing penetration depth and image quality.
  • Existing methods struggle with scattering and optical aberrations at greater depths.
  • High-fidelity visualization of subcellular structures in thick biological samples remains a challenge.

Purpose of the Study:

  • To introduce a novel imaging system, confocal² spinning-disk image scanning microscopy (C²SD-ISM), for enhanced deep-tissue super-resolution imaging.
  • To overcome the limitations of background interference and improve imaging fidelity in biological samples.
  • To achieve high-resolution imaging at greater depths than previously possible.

Main Methods:

  • Integration of a spinning-disk (SD) confocal microscope with a digital micromirror device (DMD) for sparse multifocal illumination.
  • Implementation of a dynamic pinhole array pixel reassignment (DPA-PR) algorithm for image scanning microscopy (ISM) super-resolution reconstruction.
  • Dual confocal configuration to enhance resolution and mitigate scattering background.

Main Results:

  • Achieved an imaging depth of up to 180 μm, preserving intensity distribution with increasing depth.
  • Obtained a lateral resolution of 144 nm and an axial resolution of 351 nm.
  • Demonstrated high fidelity with a 92% linear correlation between original confocal and reconstructed images, correcting for aberrations and Stokes shifts.

Conclusions:

  • The C²SD-ISM system effectively mitigates scattering background and enhances resolution for deep-tissue imaging.
  • The system offers high-fidelity super-resolution imaging with improved depth penetration and aberration correction.
  • Its versatility and scalability, including compatibility with structured illumination microscopy, make it a valuable tool for bioimaging.