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Analysis of three-dimensional objects in quantitative phase contrast microscopy: A validity study of the planar approximation for spherical particles

par Dohet-Eraly, Jérôme ;Méès, Loïc;Olivier, Thomas;Dubois, Frank ;Fournier, Corinne
Référence Proceedings of SPIE - The International Society for Optical Engineering, 11351, 113510Y
Publication Publié, 2020-08-01

Article révisé par les pairs

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Résumé :

Phase contrast microscopy is highly valuable in medicine, biology, fluid dynamics, etc., as it allows in focus observation of transparent or semi-transparent objects, which are difficult to analyze with conventional bright field microscopy. Indeed, such samples mainly affect the phase of the optical field, i.e., the shape of the wavefront, but not the light intensity. Consequently, techniques providing quantitative phase contrast in microscopy, e.g., digital holography, are suitable for transparent object characterization: assessing the thickness, or more precisely the optical thickness, of an object directly from its phase profile is a very common approximation. However, the phase profile in the object median plane is generally different from its thickness profile, as actual three-dimensional objects cause wavefront distortion. This paper discusses the validity and limitations of this approximation. The presented study considers simulated homogeneous, transparent, spherical particles. The optical field behind the particle, computed using Mie theory, is backpropagated to the object plane by means of Rayleigh-Sommerfeld propagation equation. We have shown that the approximation is better for larger spheres and, to a certain extent, for lesser refractive index difference between the object and the surrounding medium. Moreover, the error in assessing the object thickness directly from the central value of the phase profile, has been studied. Considering, for example, a siliceous sphere in oil or in air, the error increases rapidly above 5% for diameters smaller than the illumination wavelength. The impact of a slight defocus has also been studied.