This paper present a novel approach to perform the tomography of biological specimen based on Digital Holographic Microscopy (DHM). A hologram results from the interference between a reference wave and an object wave reflected from or transmitted through a sample. In the hologram, both amplitude and phase of the field transmitted through the object are registered. In DHM, the object field is recovered when the hologram is processed by a digitally computed replica of the reference wave, allowing quantitative measurement of both phase and amplitude. Phase measurements provide high accuracy optical path length measurements across the specimen along the optical axis. To proceed to a tomographic reconstruction of the refractive index of the sample based on this quantitative phase measurement, such 2-dimentionnal data must be recorded for different sample orientations covering an angle of 180° to cover all the object spatial frequencies in the reciprocal space. The representation of the data in function of the angle is known as a sinogram. The 3-dimentionnal refractive index can then be reconstructed from the sinograms by a filtered backprojection algorithm. In our system, the specimen is inserted in a glass micropipette to permit its rotation. To our knowledge, a quantitative tomography of the refractive index of a pollen cell with a resolution in the micron range is presented for the first time.

Date Published: 23 February 2006
PDF: 8 pages
Proc. SPIE 6090, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, 609008 (23 February 2006); doi: 10.1117/12.645903

Published in SPIE Proceedings Vol. 6090:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson, Editor(s)