Abstract
Oblique back-illumination microscopy (OBM) provides high resolution, sub-surface phase-gradient images from arbitrarily thick samples. We present an image formation theory for OBM and demonstrate that OBM lends itself to volumetric imaging because of its capacity for optical sectioning. In particular, OBM can provide extended depth of field (EDOF) images from single exposures, by rapidly scanning the focal plane with an electrically tunable lens. These EDOF images can be further enhanced by deconvolution. We corroborate our theory with experimental volumetric images obtained from transparent bead samples and mouse cortical brain slices.
Highlights
Phase contrast microscopy provides exquisite high-resolution images of sample morphology, without the use of sample labeling
We have recently developed a new technique called Oblique Back-illumination Microscopy (OBM) that provides microscopic resolution differential interference contrast (DIC)-like images of sub-surface sample morphology in arbitrarily thick tissue [2]
Because the light sources are off axis, the collected illumination traverses the focal plane in an oblique manner, leading to phase-gradient contrast [3,4,5]
Summary
Phase contrast microscopy provides exquisite high-resolution images of sample morphology, without the use of sample labeling. Standard phase contrast techniques, like differential interference contrast (DIC), only work in the transmission direction and cannot be used when imaging thick samples (see [1] for review). We have recently developed a new technique called Oblique Back-illumination Microscopy (OBM) that provides microscopic resolution DIC-like images of sub-surface sample morphology in arbitrarily thick tissue [2]. We illustrate the capacity of OBM to provide both fast volumetric imaging from multiple exposures and extended depth of field (EDOF) imaging from single exposures. These techniques are corroborated by experiments with transparent bead samples in scattering media and unlabeled mouse brain slices
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