RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems

Abstract

AbstractAutofocus methods used in biomicroscopy are either based on the search of an optimal focus position – which requires suspending data collection during the optimization process – or on the continuous monitoring of the position of a fiducial plane – which may not coincide with the sample itself. Here, we introduce RAPID (Rapid Autofocus via Pupil-split Image phase Detection), a method for real-time image-based focus stabilization, applicable in all wide-field microscopy systems. We demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to light-sheet microscopy. RAPID provides a universal autofocus solution for automated microscopy, and enables quantitative assays otherwise impossible in a standard microscope, such as 3D tracking of fast-moving organisms.