Abstract

Digital Holography in microscope configuration thanks to the numerical reconstruction procedure is a flexible and useful tool for analysis of biological material. We present the investigation of lipid particles growth in in-vitro mouse cell using a Digital Holographic Microscopy (DHM) employed in combination with Lateral Shear Interferometry (LSI). The optical setup is based on a Mach-Zehnder interferometer in transmission geometry. The sample cell is placed in one interferometer arm while the other one is used as a reference beam. By means of the Rayleigh-Sommerfield integral is possible to retrieve the complex object field and then to calculate the amplitude and phase of the laser light transmitted by the sample. Traditional microscopy allows to obtain amplitude contrast image only, DH, instead, enables to calculate the phase map of the complex wave that is simply related to the optical phase difference (OPD) experienced by the light when it is transmitted through the object. In this way it is possible to obtain phase contrast image that is very useful for biological materials that often present low amplitude contrast for quantitative amplitude image. The main difficulty of this technique is to remove the optical aberrations produced by the optical setup components. Several methods have been proposed, such as subtraction of a reference phase map (without sample) [1] or numerical multiplication of a parametric lens [2], We propose a fast and effective solution of this problem based on LSI. We digitally introduce a lateral shear of one pixel in x and y directions calculating the phase difference Δϕ_x,y, between the actual phase map and its sheared replica in both directions. Δϕ_x,y include a linear term due to defocus aberration and the object phase difference. The linear term can be easily eliminated and sample phase map retrieved by numerical integration. This technique allows to obtain the correct phase contrast image removing optical aberration, avoiding unwrapping problems.

© 2007 SPIE