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

Ludovico Silvestri, Marie Caroline Muellenbroich, Irene Costantini, Antonino Paolo Di Giovanna, Leonardo Sacconi, Francesco Saverio Pavone
doi: https://doi.org/10.1101/170555
Ludovico Silvestri
National Institute of Optics (INO-CNR);
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  • For correspondence: silvestri@lens.unifi.it
Marie Caroline Muellenbroich
National Institute of Optics (INO-CNR);
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Irene Costantini
European Laboratory for Non-linear Spectroscopy;
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Antonino Paolo Di Giovanna
European Laboratory for Non-linear Spectroscopy;
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Leonardo Sacconi
National Institute of Optics (INO-CNR);
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Francesco Saverio Pavone
University of Florence
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Abstract

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.

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Posted July 31, 2017.
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RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems
Ludovico Silvestri, Marie Caroline Muellenbroich, Irene Costantini, Antonino Paolo Di Giovanna, Leonardo Sacconi, Francesco Saverio Pavone
bioRxiv 170555; doi: https://doi.org/10.1101/170555
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RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems
Ludovico Silvestri, Marie Caroline Muellenbroich, Irene Costantini, Antonino Paolo Di Giovanna, Leonardo Sacconi, Francesco Saverio Pavone
bioRxiv 170555; doi: https://doi.org/10.1101/170555

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