Multi-wavelength digital holographic microscopy for sub-micron topography of reflecting specimens

The study of the internal structures of specimens has a great importance in life and materials sciences. The principle of optical diffraction tomography (ODT) consists in recording the complex wave diffracted by an object, while changing the k vector of the illuminating wave. This way, the frequency domain of the specimen is scanned, allowing reconstructing the scattering potential of the sample in the spatial domain. This work presents a method for sub-micron tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording at different wavelengths equally separated in the k-domain, of the interference between an off-axis reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A charged coupled device (CCD) camera records consecutively the holograms, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefronts. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and a destructive one in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables to scan the object in depth. For the presented simulations and experiments, twenty wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices of 725 nm thick.