Artifacts in Biological Confocal and Non-linear Microscopy

Abstract

Conventional histological sectioning and staining techniques produce tissue sections, by large, independent from each other (e.g. the staining intensity depends on the total stainable substance within the section, but not influence by the section above or below it). Although the hardness of the structure in a given section may influence the quality of the following sections, never the less, each physical section can be generally regarded as an independent sampling event. However, the image intensity and relative contrast in an optical section obtained from confocal or non-linear optical microscopy varies depending on the position of the section where it was obtained. Therefore, in sampling terms, optical sections are dependent sampling events. Artifacts caused by the geometry and optical properties (e.g. absorption, scattering, refractive index) of objects within a specimen can interfere the interpretation of the result and hinder the usefulness of three-dimension reconstruction. Figure 1 demonstrates intensity variation in confocal and multi-photon micrographs due to the geometry of the specimen. Depends on the absorption coefficient and path length of the illumination and the fluorescecent light, a solid spherical object produced a 3D data set, when reconstructed, resemble an inverted Chinese rice bowl. Figure 2 shows a 3D reconstruction of an Arabidopsis thaliana mesophyll protoplast; note only half of the spherical cell facing the illumination is imaged as shown in the cut-off view in FIG 3.