Submicrometer optical tomography by multiple-wavelength digital holographic microscopy

We present a method for submicrometer 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, in off-axis geometry, of the interference between a reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A CCD camera records the holograms consecutively, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefront. 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 destructive addition 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 one to scan the object in depth. For the presented simulations and experiments, 20 wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices 725 nm thick.