Quantitative phase imaging (QPI) is an important tool in biomedicine that allows for the microscopic investigation of live cells and other thin, transparent samples. Importantly, this technology yields access to the cellular and sub-cellular structure and activity at nanometer scales without labels or dyes. Despite this unparalleled ability, QPI's restriction to relatively thin samples severely hinders its versatility and overall utility in biomedicine. Here we overcome this significant limitation of QPI to enable the same rich level of quantitative detail in thick scattering samples. We achieve this by first illuminating the sample in an epi-mode configuration and using multiple scattering within the sample-a hindrance to conventional transmission imaging used in QPI-as a source of transmissive illumination from within. Second, we quantify phase via deconvolution by modeling the transfer function of the system based on the ensemble average angular distribution of light illuminating the sample at the focal plane. This technique packages the quantitative, real-time sub-cellular imaging capabilities of QPI into a flexible configuration, opening the door for truly non-invasive, label-free, tomographic quantitative phase imaging of unaltered thick, scattering specimens. Images of controlled scattering phantoms, blood in collection bags, cerebral organoids and freshly excised whole mouse brains are presented to validate the approach.

Three-dimensional (3D) refractive index (RI) tomography has recently become an exciting new tool for biological studies. However, its limitation to (1) thin samples resulting from a need of transmissive illumination and (2) small fields of view (typically ∼ 50 µ m × 50 µ m ) has hindered its utility in broader biomedical applications. In this work, we demonstrate 3D RI tomography with a large field of view in opaque, arbitrarily thick scattering samples (unsuitable for imaging with conventional transmissive tomographic techniques) with a penetration depth of ca. one mean free scattering path length ( ∼ 100 µ m in tissue) using a simple, low-cost microscope system with epi-illumination. This approach leverages a solution to the inverse scattering problem via the general non-paraxial 3D optical transfer function of our quantitative oblique back-illumination microscopy (qOBM) optical system. A theoretical analysis is presented along with simulations and experimental validations using polystyrene beads, and rat and human thick brain tissues. This work has significant implications for the investigation of optically thick, semi-infinite samples in a non-invasive and label-free manner. This unique 3D qOBM approach can extend the utility of 3D RI tomography for translational and clinical medicine.

There is currently no low-cost method to quantitatively assess the contents of a blood bag without breaching the bag and potentially damaging the sample. Towards this end, we adapt oblique back-illumination microscopy (OBM) to rapidly, inexpensively, and non-invasively screen blood bags for red blood cell (RBC) morphology and white blood cell (WBC) count. OBM has been recently introduced as a tomographic technique that produces high-resolution wide-field images based on phase-gradient and transmission. Here we modify this technique to include illumination at dual wavelengths to facilitate spectral analysis for cell classification. Further, we apply a modified 2D Hilbert transform to recover the phase information from the phase-gradient images for facile cell segmentation. Blood cells are classified as WBCs and RBCs, and counted based on shape, absorption spectrum, and phase profile using an automated algorithm. This work has important implications for the non-invasive assessment of (1) cell viability in storage bags for transfusion applications and (2) suitability of a cord blood collection bag for stem cell therapy applications.