We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.

For what we believe to be the first time, digital holographic microscopy is applied to perform optical diffraction tomography of a pollen grain. Transmission phase images with nanometric axial accuracy are numerically reconstructed from holograms acquired for different orientations of the rotating sample; then the three-dimensional refractive index spatial distribution is computed by inverse radon transform. A precision of 0.01 for the refractive index estimation and a spatial resolution in the micrometer range are demonstrated.

We present a procedure that compensates for phase aberrations in digital holographic microscopy by computing a polynomial phase mask directly from the hologram. The phase-mask parameters are computed automatically without knowledge of physical values such as wave vectors, focal lengths, or distances. This method enables one to reconstruct correct and accurate phase distributions, even in the presence of strong and high-order aberrations. Examples of applications are shown for microlens imaging and for compensating for the deformations associated with a tilted thick plate. Finally we show that this method allows compensation for the curvature of the specimen, revealing its surface defects and roughness. Examples of applications are shown for microlenses and metallic sphere imaging.

The concept of numerical parametric lenses (NPL) is introduced to achieve wavefront reconstruction in digital holography. It is shown that operations usually performed by optical components and described in ray geometrical optics, such as image shifting, magnification, and especially complete aberration compensation (phase aberrations and image distortion), can be mimicked by numerical computation of a NPL. Furthermore, we demonstrate that automatic one-dimensional or two-dimensional fitting procedures allow adjustment of the NPL parameters as expressed in terms of standard or Zernike polynomial coefficients. These coefficients can provide a quantitative evaluation of the aberrations generated by the specimen. Demonstration is given of the reconstruction of the topology of a microlens.

This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of π, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 μm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.