Abstract: A well-known distortion of objects in three-dimensional microscopy manifests itself as an elongation in the axial direction. Authors such as Visser and Hell have seemingly contradicted one another on the cause as well as the magnitude of the effect. We have examined these theories and performed simulations and experimental measurements to better understand the nature of the effect. We simulate point spread functions (based on the work of Gibson) taking into account the various refractive indices involved as well as the magnification, the numerical aperture, the working distance of the objective, the depth of the object under the coverslip, and the object's size. We measure the axial and lateral dimensions of digitized images of microspheres that have been 'acquired' using a simulated point spread function that changes as the depth of the object changes. These simulations are done for conventional (optical sectioning) microscopy as well as for confocal microscopy. Further, we have performed experimental measurements on real microspheres on a conventional microscope to relate theory, simulation, and practice. Our measurements and simulations show that (1) the object's size, (2) its depth under the coverslip, (3) the refractive index mismatch between the immersion fluid (n$-immersion$/) and embedding material for the object (n$-embedded$/), and (4) the NA of the lens play a pivotal role in the effect. !13
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