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 traveled 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 medium with a higher RI than that of the sample) or compressed images (if the objective is immersed in a medium with a lower RI than that of 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.

当从球面透镜的不同点进入的光线没有聚焦到光轴的同一点时，就会出现球面像差 (SA)。荧光显微镜镜头元件内部发生的 SA 已被很好地理解和纠正。然而，当光穿过折射率 (RI) 不匹配物质的界面（即浸没介质的 RI 与样品的 RI 之间的差异）时，也会引发 SA。由于 RI 不匹配而导致的 SA 对成像有许多有害影响。对于 3D 成像来说，最重要的也许是图像平面在样本中移动的距离不等于z堆栈采集期间物镜（或平台）移动的距离。这种沿z轴的不均匀平移会产生人为拉长的图像（如果物镜浸入 RI 高于样品的介质中）或压缩图像（如果物镜浸入 RI 较低的介质中）比样本）并改变最佳轴向采样率。在本教程中，我们将描述发生这种失真的原因、它如何影响定量测量和轴向分辨率，以及如何避免 SA 从而防止图像失真。此外，本教程旨在更好地告知研究人员如何纠正 RI 不匹配引起的轴向变形，并提供实用的基于 ImageJ/Fiji 的工具来减少体积测量误差和轴向分辨率损失的发生率。