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We developed a dual-view oblique plane microscope (dOPM) for high-resolution imaging of thick, cleared tissues. This advanced microscope achieves detailed multicolour volumetric imaging across various biological samples.

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Area of Science:

  • Biomedical Optics
  • Microscopy
  • Biophotonics

Background:

  • Imaging thick, optically cleared biological tissues presents challenges in maintaining spatial resolution and optical quality.
  • Existing microscopy techniques often struggle with refractive index mismatches and limited penetration depth in dense samples.

Purpose of the Study:

  • To introduce a novel dual-view oblique plane microscope (dOPM) for high-resolution volumetric imaging of thick, optically cleared biological samples.
  • To characterize the dOPM's performance, particularly its remote refocusing capabilities and spatial resolution across varying refractive indices.
  • To demonstrate the dOPM's versatility by imaging diverse biological specimens, including cancer tissues and whole organisms.

Main Methods:

  • Development of a custom remote refocusing relay using stock optics for refractive index-matched samples.
  • Spatial resolution characterization using fluorescent bead phantoms (RI 1.4-1.5) to determine point spread function (PSF) FWHM.
  • Application of tiled acquisition, image stitching, and multi-view image fusion for comprehensive volumetric reconstruction.
  • Imaging of optically cleared mouse tissues (ovarian cancer, bone marrow, colon) and a whole *Drosophila melanogaster* brain.

Main Results:

  • Achieved lateral and axial resolutions of ~0.5 µm and ~1 µm, respectively, in refractive index-matched samples.
  • Demonstrated minimal resolution degradation over an axial range >250 µm.
  • Showcased effective compensation for refractive index variations up to n=1.5 using objective correction collars.
  • Successfully performed multicolour volumetric imaging of mm-wide tissue samples and a whole fruit fly brain.

Conclusions:

  • The dOPM provides a robust platform for fast, high-resolution, multicolour volumetric imaging of large biological samples.
  • The system integrates advanced optical techniques on a commercial microscope frame, offering accessibility and broad applicability.
  • This technology enables detailed investigation of complex biological structures across multiple scales, advancing research in various life science fields.