Tutorial: Avoiding and Correcting Sample-Induced Spherical Aberration Artifacts in 3D Fluorescence Microscopy

Abstract

Spherical Aberration (SA) occurs when light rays entering at different points of a spherical lens are not focused to the same point of the optical axis. SA that occurs inside the lens elements of a fluorescence microscope is well understood and corrected for. However, SA is also induced when light passes through an interface of refractive index (RI) mismatched substances (i.e. a discrepancy between the RI of the immersion medium and the RI of the sample). SA due to RI mismatches has many deleterious effects on imaging. Perhaps most important for 3D imaging is that the distance the image plane moves in a sample is not equivalent to the distance travelled by an objective (or stage) during Z-stack acquisition. This non-uniform translation along the Z-axis gives rise to artifactually elongated images (if the objective is immersed in a higher RI medium than the sample) or compressed images (if the objective is immersed in a lower RI medium than the sample) and alters the optimal axial sampling rate. In this Tutorial, we describe why this distortion occurs, how it impacts quantitative measurements and axial resolution and what can be done to avoid SA and thereby prevent distorted images. In addition, this tutorial aims to better inform researchers of how to correct RI mismatch-induced axial distortions and provides a practical ImageJ/Fiji-based tool to reduce the prevalence of volumetric measurement errors and lost axial resolution.