Development of micro-optical projection tomography for 3D analysis of single cells

Abstract

The Micro-Optical Projection Tomographic Microscope (μOPTM) is an instrument that is being developed for three-dimensional (3D) imaging of cells and subcellular components. The target application for the μOPTM is the early detection of lung cancer by revealing the complex 3D information about chromatin redistribution in the nucleus. The µOPTM employs a scanning objective lens (100x, N.A.=1.25) to create an extended depth-of-field image, similar to a shadowgram or projection, that we call a pseudo-projection. A large number of pseudo-projections (90+) are acquired, from which a tomographically reconstructed 3D image is computed using a filtered backprojection algorithm. The prototype μOPTM uses a single objective lens, so the object (cell) must be rotated for each new pseudo-projection. A custom microtube stage minimizes the lateral and axial motion of the sample tube during scanning and rotation so that registration between successive pseudo-projections is maintained. Image processing methods are used to correct any remaining registration errors. The media inside and outside the tube are refractive index-matched to each other and to the tube (Δnavg < 0.02). The index-matched materials are pressed between two flat parallel windows, providing a nearly distortion-free image. Custom phantoms using microspheres have been constructed and images of these 3D test targets acquired. The minimum resolvable feature size is better than 3 microns. The first 3D image of a cell using μOPTM is also shown.