Label-free 3D imaging of weakly absorbing samples using spatially-incoherent annular illumination microscopy

Abstract

In this paper, we present a simple but effective label-free three dimensional (3D) microscopy for weakly absorbing samples. The proposed technique employs spatially-incoherent annular illumination to enhance absorption contrast of in-focus images and reduce phase contrast. We also employ mechanical scanning along axial direction to acquire a volume of the sample images. A 3D gradient operation is further adopted to remove the background with defocused shadows caused by oblique illumination. As such, a sequence of background-free sectioning images is acquired. The 3D gradient operation results in that only the structural edges of the weakly absorbing sample are visible in the images. We can therefore reconstruct the 3D skeleton structure of the sample from the sectioning image sequence. A label-free diatom is used to verify our technique experimentally. The 3D skeleton structure of the diatom is reconstructed and presented. The proposed technique would find applications in various fields, such as life science, materials science, etc.