Label-Free Quantitative Phase Imaging Reveals Spatial Heterogeneity of Extracellular Vesicles in Select Colon Disorders

Three-dimensional (3D) imaging and quantitative analysis of extracellular vesicles (EVs) remain largely unexplored, mainly because of limitations in detection techniques. In this study, EVs from patients diagnosed with colorectal cancer (CRC) and ulcerative colitis were examined. To investigate the spatial heterogeneity and 3D refractive index (RI) distribution of single EVs, a label-free digital holographic tomography technique was used at a submicrometer spatial resolution. The presented image-processing algorithms were used in quantitative analysis with digital staining and 3D visualization, the determination of the EV size distribution and extraction of fractions with different RIs. Reconstructed 3D RI distributions revealed variations in the spatial heterogeneity of EVs related to tissue specificity, such as CRC, normal colonic mucosa, and ulcerative colitis, as well as the isolation procedures used. The RI values of EVs isolated from solid tissues of frozen CRC samples were also dependent on the tumor grade and cancer cell proliferation. The simultaneous examination of cell culture models confirmed the association of the RI of EVs with the tumor grade. 3D-RI data analysis generates new perspectives with the optical, contact-free, label-free examination of the individual EVs. Depending on the specific tissue and isolation method, EVs exhibit significant spatial heterogeneity. The optical parameters of single EVs enabled their classification into two unique subgroups with different RI values.