Optical projection microtomography of transparent objects

摘要

We report first results in the development of a diffractive optical microtomography instrument for imaging transparent/semi-transparent biological samples without staining and sectioning. A brightfield transmission microscope was modified to form a Mach-Zehnder interferometer that was used to generate phase-shifted holograms recorded in image plane. Transparent/semi-transparent objects mixed with an index matching medium were inserted into a microcapillary and holograms of these objects were taken under different view angles by rotating the microcapillary. Precise rotation of the microcapillary was accomplished by clipping the microcapillary in a precisely machined V-groove, a system that when combined with software correction of the object centre achieved a precision of object positioning on the order of a micrometer. In this study, the observed objects were considered to be weakly diffracting and reconstructed by projection tomography of the phases of their measured scattered fields. The three-dimensional distribution of the refractive index was obtained by backprojecting the phases. Refractive index distributions are shown for a glass bead and a pollen grain. The measured difference between the refractive index of the glass bead and the microcapillary was within ±0.01. An isotropic spatial-resolution of the instrument in the micrometer range was obtained with an objective having a numerical aperture of 0.4.