Our group at the BC Cancer Research Centre has developed an optical computed-tomography microscope for three-dimensional
(3-D) imaging and quantitative analysis of absorption-stained biological samples. The device uses non-ionizing
optical radiation for illumination and computed-tomography for 3-D image reconstruction. The optical system consists of a
light source, two high-numerical aperture (NA) objective lenses, a sample stage, a CCD detector, and a spatial light
modulator that is used for computer-controlled sample illumination. Projections of a specimen are measured at a set of
appointed angles within the range of 0 less than or equal to angle less than or equal to 135, which is limited by the NA of the objective. The reconstruction
algorithm developed to solve the limited-angle problem employs the reconstruction from a transform-based method as an
initial guess for the following iterative reconstruction. 3-D microscopic images of quantitatively absorption-stained cells have
been generated with the resolution on the order of 1 micron. Visualization of 3-D tissue structure and architectural and
morphological features that can be extracted from the volume to provide pathologists with additional information for
We present our results on development of algorithms for image reconstruction in optical computed-tomography microscopy. The optical computed-tomography microscope with light modulation, recently developed by our group at the BC Cancer Research Centre is a novel imaging device for three-dimensional visualization and quantitative analysis of absorption-stained biological samples. The angles for projection in the system are limited to the range of 0 ≤ φ ≤ 135° by the numerical aperture of the illumination objective. For the limited-angle tomography problem we have developed several reconstruction algorithms. One algorithm is based on the Radon transformation and assumes parallel ray projections. In order to compensate for limited data, several reconstructions are generated from several sets of projections of a specimen, acquired at different orientations of parallel-ray light scanning. The reconstructions are combined together using a vote criteria to create a final volume. Another reconstruction algorithm developed by the group employs both transform-based and iterative methods to address the limited-angle reconstruction problem. In this algorithm the transform-based method is used as an initial starting point for the following iterative reconstruction. A feedback correction of the reconstruction image is made on each iteration step. The method enables to incorporate previously known information about the object into the reconstruction process. The algorithm improves reconstruction accuracy at a reasonable computational cost and programming commitment. Three-dimensional microscopic images of quantitatively absorption-stained cells have been reconstructed with the resolution better than 6 microns.
We present our recent results on the development of three-dimensional (3-D) optical computed- tomography microscope. The instrument is a novel imaging device for the 3-D visualization and quantitative analysis of absorption-stained biological samples. The first instrument developed by our group at the BC Cancer Research Centre used a digital micromirror device (DMD) as a spatial light modulator to control the angles of illumination. This new embodiment employs an optical scanner instead of the DMD. The optical scanner is placed in the illumination path of the microscope system, conjugate to the field plane. The optical system includes also two high numerical aperture objective lenses, a sample stage, a light source, and a CCD camera. Projections are acquired by illuminating a specimen at a number of selected angles within the numerical aperture of the objective (0 < φ < 135°). A new reconstruction algorithm that employs both transform-based and iterative methods is developed to address the limited-angle reconstruction problem. A transform-based reconstruction is used as an initial starting point for the following iterative reconstruction. A feedback correction of the reconstructed image is made on each iteration step. The algorithm enables to incorporate previously known information about the object into the reconstruction process, and improves the reconstruction accuracy. Microscopic 3-D volume reconstructions of quantitatively absorption-stained cells have been generated. The system enables one to look at multiple optical levels of a specimen, and at more natural tissue architecture, including intact cells. Axial and lateral resolutions were measured to be better than 6 microns.
A computed-tomography microscope using a digital spatial light modulator has been developed and has demonstrated cytological and histological three-dimensional (3-D) image acquisition. The system consists of two high NA objective lenses, a Digital Micromirror Device (DMD) placed conjugate to the back pupil plane of the illumination objective, a sample stage, a light source, and a CCD detector. Each DMD micromirror can control the illumination of a specific angle. A single 3-D reconstruction is obtained from parallel ray projections acquired by changing the polar angle of illumination -phi_max < phi < phi_max while holding the azimuthal angle theta constant. The polar angle is limited by the NA of the objective. To compensate for an incompleteness of information due to the limited polar angles, several reconstructions acquired at multiple azimuthal angles are combined to create a final reconstruction. A reconstruction algorithm was developed using simulation software based on the 3-D Radon transformation and 3-D synthetic objects. Microscopic 3-D volume reconstructions of quantitatively absorption-stained cells have been demonstrated. 3-D reconstructed images enables the analysis of cell morphology and tissue architecture, as well as virtual two-dimensional slices with the distance between slices of 0.3 μm.