PT  - JOURNAL ARTICLE
AU  - L. Silvestri
AU  - M. C. Müllenbroich
AU  - I. Costantini
AU  - A. P. Di Giovanna
AU  - L. Sacconi
AU  - F. S. Pavone
TI  - RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems
AID  - 10.1101/170555
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 170555
4099  - http://biorxiv.org/content/early/2017/07/31/170555.short
4100  - http://biorxiv.org/content/early/2017/07/31/170555.full
AB  - Autofocus 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.