Multiple wavelength digital holographic microscopy for the three dimensional imaging of biostructures

The Institute of Cybernetics" E Caianiello" has developed a digital holographic microscope for quantitative imaging of biostructures. A digital holographic microscope relies on the classical principle of holography except that the hologram is not recorded by a traditional film, but by means of a CCD array sensor camera, typically a sensor of 1024 x 1024 pixel of 5-10 micron size. Each pixel records the intensity of the resulting interference pattern. The hologram is recorded and digitized into a numerical matrix, which is processed to obtain the three-dimensional distribution of the object under investigation. The numerical reconstruction of a digitized hologram represents an important task [1] of the microscope, since it employs specific image processing algorithms to determine the phase image of the microstructure, in addition to the amplitude image obtained by a standard microscope. In order to apply the technique to the reconstruction of three-dimensional images of biostructures, it has been employed three laser sources in the visible region to obtain the corresponding three phase images, thus improving the spatial resolution and the measurement accuracy compared to the single wavelength technique. This technique has been employed to obtain phase images of chromosomal and cellular structures, that quantify the optical path length of the laser radiation through the structure under investigation

[1] "Quantitative phase-contrast digital holography method for the numerical reconstruction of images, and relevant apparatus", P. Ferraro, Domenico Alfieri, Sergio De Nicola, Andrea Finizio, Giovanni Pierattini, WIPO Patent Application WO/2007/122655- PCT/IT2007/000224 (03/26/2007)