Multiscale label-free volumetric holographic histopathology of thick-tissue slides with subcellular resolution

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.

Long-term label-free assessments of individual bacteria using three-dimensional quantitative phase imaging and hydrogel-based immobilization

“…Traditional AST utilizes populational information of bacteria when measuring their susceptibility to antibiotics 45 . Our protocol can be implemented for not only population-based AST 24 , but also for assessing the drug susceptibility of individual bacterium, which has been indicated as a rapid alternative to the conventional time-consuming approaches 28 . A commercialized label-free individual bacteria AST method evaluates samples in an agarose-based environment using bright-field microscopy.…”

“…QPI enables label-free, high-contrast images by measuring the optical phase delay of scattered light 16 . By enabling investigations at the single-cell level, QPI has been utilized to study blood cells 17 , 18 , cancer cells 19 – 21 , SARS-CoV-2 virus 22 , tissues 23 , 24 , and bacteria 13 , 25 , 26 . Due to its high-resolution, label-free, quantitative imaging capability, 2D QPI techniques have been utilized for the study of bacteria.…”

Label-free histological analysis of retrieved thrombi in acute ischemic stroke using optical diffraction tomography and deep learning

“…To obtain whole-slide RI tomograms, multiple tomograms were stitched following the measurements in separate locations [33]. A phase-correlation algorithm [33, 34] was used to determine the relative position between overlapping tomogram tiles, and stitch them into tomograms. By automatically stitching the scanned tomograms, the ODT setup achieved a millimeter-scale FOV (10 mm 2 ).…”

Roadmap on Digital Holography-Based Quantitative Phase Imaging

“…This is because of the limited space-bandwidth product of QPI [18] and the considerable computation necessary during the reconstruction process. To image a larger area, tiled images can be measured and stitched [98]. However, sequential imaging and stitching are time-consuming, and handling large-sized image data consumes considerable computational power.…”

“…The 3D QPI is explored with a variety of methods, with the limited-angle ODT leading the way in terms of accuracy of the reconstructions and commercialization efforts in the area of biomedicine. With the rise in popularity of the 3D QPI techniques, including multiscale and multimodal approaches [98,151,152], as well as the use of machine learning to facilitate both reconstruction process and data analysis [67,155,156], it is of utmost importance to measure and consider the uncertainty of the underlying data. It is clear that cross-referencing the results between different techniques, optomechatronic systems, and laboratories, as well as the future databases, has immense value and can only be achieved with proper metrology.…”