Although 3D imaging techniques are useful for studying thick biological samples, the challenge to achieve both high-speed acquisition and high resolution of 3D images persists. Intensity diffraction tomography (IDT), a label-free phase tomography technique, can help overcome these limitations in 3D imaging. Researchers at Boston University (BU) developed two new IDT methods — annular IDT (aIDT) and multiplexed IDT (mIDT) — to boost the image acquisition speed in 3D imaging techniques. Annular IDT uses an LED ring that matches the objective’s numerical aperture (NA). Multiplexed IDT uses multiple LEDs that illuminate the sample simultaneously. Both IDT methods improve 3D imaging speed enough to enable visualization of dynamic biological samples. The researchers found that existing IDT reconstruction algorithms did not work well with the aIDT and mIDT techniques, due to the use of high-NA objectives. To circumvent this issue and optimize the aIDT and mIDT techniques, the researchers developed a new algorithm. The new algorithm uses a multiple scattering model based on the split-step, nonparaxial (SSNP) method, which was originally developed to overcome similar limitations in optical diffraction tomography. The algorithm captures complex 3D light scattering information from live specimens, recovering the 3D refractive index distribution of biological samples that exhibit multiple types of light scattering. The researchers combined the new algorithm with IDT to characterize thick biological samples. They used the IDT reconstruction algorithm with aIDT to image buccal epithelial cells, and could easily discriminate between cells at different depths, reconstruct the cell boundaries and membrane, and visualize native bacteria around the cells. The researchers also used the algorithm with mIDT to image thick, multiscattering, live C. elegans embryos. The resulting reconstructed images showed details of how the worms were folded, and the single-depth cross-section showed the morphological details of the cells’ outline, the buccal cavity, and the tail of the worm. To perform IDT, a programmable LED array can be added easily to a standard microscope. Overall, the BU team’s experiments showed that by extending the SSNP method to IDT, it was possible to achieve high-quality 3D images with a large field of view. Another approach that is used for 3D imaging of biological samples is quantitative phase imaging (QPI), a technique that provides quantitative, volumetric refractive index reconstructions of unlabeled biological samples from intensity-only measurements. BU researcher Jiabei Zhu, who led the team that developed aIDT, mIDT, and the IDT reconstruction algorithm, said, “3D quantitative phase imaging (QPI) has superior features for various applications in the field of biomedical imaging. As a label-free technique, QPI can image transparent living organisms and cells without exogenous contrast agents and dyes which induce phototoxic effects damaging the sample. “Compared with traditional phase-contrast and differential interference contrast microscopy, QPI not only provides high-contrast morphological information but gives quantitative phase information as well,” Zhu said. “Specifically, 3D QPI can provide high-resolution 3D refractive index distribution inside the samples. This valuable information can facilitate the research on hematology, neurology, and immunology, helping the diagnosis of disease and infection.” Zhu presented the BU researchers’ work in the area of IDT at the Optica Imaging Congress, Aug. 14-17, in Boston.