Abstract
Histopathology relies upon the staining and sectioning of biological tissues, which can be laborious and may cause artifacts and distort tissues. We develop label-free volumetric imaging of thick-tissue slides, exploiting refractive index distributions as intrinsic imaging contrast. The present method systematically exploits label-free quantitative phase imaging techniques, volumetric reconstruction of intrinsic refractive index distributions in tissues, and numerical algorithms for the seamless stitching of multiple three-dimensional tomograms and for reducing scattering-induced image distortion. We demonstrated label-free volumetric imaging of thick tissues with the field of view of 2 mm × 1.75 mm × 0.2 mm with a spatial resolution of 170 nm × 170 nm × 1400 nm. The number of optical modes, calculated as the reconstructed volume divided by the size of the point spread function, was ∼20 giga voxels. We have also demonstrated that different tumor types and a variety of precursor lesions and pathologies can be visualized with the present method.
Highlights
Microscopic assessment of biopsied and resected tissues is central to understanding the underlying pathophysiology and clinical states of many patients
We addressed the issues of limited FoV and image degradation by constructing a longworking-distance Optical diffraction tomography (ODT) optical setup and developing a robust multiscale ODT reconstruction and stitching algorithm, which consider optical aberration due to thick tissues
The system was based on a Mach–Zehnder interferometer, which was equipped with a long-working-distance objective lens for imaging thick tissues, an automated sample stage for raster-scanning, and a digital micromirror device (DMD; DLPLCR6500EVM, Texas Instrument) for illumination beam control
Summary
Microscopic assessment of biopsied and resected tissues is central to understanding the underlying pathophysiology and clinical states of many patients. From transmitted-light holographic measurements at various angles, similar to x-ray computed tomography, ODT reconstructs the 3D refractive index (RI) distribution of biological cells.[19,20] Because RI values depend on the number of intracellular biomolecules, including proteins and lipids, ODT allows label-free quantitative 3D morphological mapping of biological specimens and has been widely utilized to advance our understanding of the physiology of various live cells.[21,22,23,24,25] several technical issues, such as a small holographic field retrieval field-of-view (FoV) and image degradation due to multiple light scattering, have hindered 3D RI-based histopathological analyses When using this algorithm, tissue slices with thickness of up 100 μm could be reconstructed with high contrast. The experimental setup enabled mesoscopic imaging of various pathologic tissues over a millimeter-scale FoV with submicrometer resolution
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