Three-dimensional (3D) fluorescence microscopy in general requires axial scanning to capture images of a sample at different planes. Here we demonstrate that a deep convolutional neural network can be trained to virtually refocus a 2D fluorescence image onto user-defined 3D surfaces within the sample volume. With this data-driven computational microscopy framework, we imaged the neuron activity of a Caenorhabditis elegans worm in 3D using a time-sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field of the microscope by 20-fold without any axial scanning, additional hardware, or a trade-off of imaging resolution or speed. Furthermore, we demonstrate that this learning-based approach can correct for sample drift, tilt, and other image aberrations, all digitally performed after the acquisition of a single fluorescence image. This unique framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. This deep learning-based 3D image refocusing method might be transformative for imaging and tracking of 3D biological samples, especially over extended periods of time, mitigating photo-toxicity, sample drift, aberration and defocusing related challenges associated with standard 3D fluorescence microscopy techniques. Text:Three-dimensional (3D) fluorescence microscopic imaging is essential for biomedical and physical sciences as well as engineering, covering various applications 1-7 . Despite its broad

Digital holographic microscopy enables the 3D reconstruction of volumetric samples from a single-snapshot hologram. However, unlike a conventional bright-field microscopy image, the quality of holographic reconstructions is compromised by interference fringes as a result of twin images and out-of-plane objects. Here, we demonstrate that cross-modality deep learning using a generative adversarial network (GAN) can endow holographic images of a sample volume with bright-field microscopy contrast, combining the volumetric imaging capability of holography with the speckle- and artifact-free image contrast of incoherent bright-field microscopy. We illustrate the performance of this “bright-field holography” method through the snapshot imaging of bioaerosols distributed in 3D, matching the artifact-free image contrast and axial sectioning performance of a high-NA bright-field microscope. This data-driven deep-learning-based imaging method bridges the contrast gap between coherent and incoherent imaging, and enables the snapshot 3D imaging of objects with bright-field contrast from a single hologram, benefiting from the wave-propagation framework of holography.

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