RT Journal Article SR Electronic T1 Single-shot autofocus microscopy using deep learning JF bioRxiv FD Cold Spring Harbor Laboratory SP 587485 DO 10.1101/587485 A1 Henry Pinkard A1 Zachary Phillips A1 Arman Babakhani A1 Daniel A. Fletcher A1 Laura Waller YR 2019 UL http://biorxiv.org/content/early/2019/03/23/587485.abstract AB Maintaining an in-focus image over long time scales is an essential and non-trivial task for a variety of microscopic imaging applications. Here, we present an autofocusing method that is inexpensive, fast, and robust. It requires only the addition of one or a few off-axis LEDs to a conventional transmitted light microscope. Defocus distance can be estimated and corrected based on a single image under this LED illumination using a neural network that is small enough to be trained on a desktop CPU in a few hours. In this work, we detail the procedure for generating data and training such a network, explore practical limits, and describe relevant design principles governing the illumination source and network architecture.