RAPID Autofocusing in Light-Sheet Microscopy: An Implementation Guide

In light-sheet microscopy, the excitation and detection paths are uncoupled and need to be aligned independently to ensure that the sheet of light lies in the focal plane of the detection objective. However, the presence of the specimen itself can break this alignment, frustrating the very principle of this technique and introducing significant blur. RAPID (Rapid Autofocus via Pupil-split Image phase Detection) has been reported as an effective method to correct sample-induced defocusing without introducing overhead optimization time. Here, I discuss some practical aspects on the implementation of this method, with the aim of providing a guide for the researchers interested in integrating this system within their light-sheet apparatus.