Characterization of cell-induced astigmatism in high-resolution imaging

Abstract

High-resolution and super-resolution techniques become more frequently used in thick, inhomogeneous samples. In particular for imaging life cells and tissue in which one wishes to observe a biological process at minimal interference and in the natural environment, sample inhomogeneities are unavoidable. Yet sample-inhomogeneities are paralleled by refractive index variations, for example between the cell organelles and the surrounding medium, that will result in the refraction of light, and therefore lead to sample-induced astigmatism. Astigmatism in turn will result in positional inaccuracies of observations that are at the heart of all super-resolution techniques. Here we introduce a simple model and define a figure-of-merit that allows one to quickly assess the importance of astigmatism for a given experimental setting. We found that astigmatism caused by the cell’s nucleus can easily lead to aberrations up to hundreds of nanometers, well beyond the accuracy of all super-resolution techniques. The astigmatism generated by small objects, like bacteria or vesicles, appear to be small enough to be of any significance in typical super-resolution experimentation.